Compositions and methods for non-targeted activation of endogenous genes

ABSTRACT

The present invention is directed generally to activating gene expression or causing over-expression of a gene by recombination methods in situ. The invention also is directed generally to methods for expressing an endogenous gene in a cell at levels higher than those normally found in the cell. In one embodiment of the invention, expression of an endogenous gene is activated or increased following integration into the cell, by non-homologous or illegitimate recombination, of a regulatory sequence that activates expression of the gene. In another embodiment, the expression of the endogenous gene may be further increased by co-integration of one or more amplifiable markers, and selecting for increased copies of the one or more ampliflable markers located on the integrated vector. In another embodiment, the invention is directed to activation of endogenous genes by non-targeted integration of specialized activation vectors, which are provided by the invention, into the genome of a host cell. The invention also provides methods for the identification, activation, isolation, and/or expression of genes undiscoverable by current methods since no target sequence is necessary for integration. The invention also provides methods for isolation of nucleic acid molecules (particularly cDNA molecules) encoding a variety of proteins, including transmembrane proteins, and for isolation of cells expressing such transmembrane proteins which maybe heterologous transmembrane proteins. The invention also is directed to isolated genes, gene products, nucleic acid molecules, to compositions comprising such genes, gene products and nucleic acid molecules, and to vectors and host cells comprising such genes and nucleic acid molecules, that may be used in a variety of therapeutic and diagnostic applications. Thus, by the present invention, endogenous genes, including those associated with human disease and development, may be activated and isolated without prior knowledge of the sequence, structure, function, or expression profile of the genes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 09/276,820, filed Mar. 26, 1999, entitled “COMPOSITIONS AND METHODSFOR NON-TARGETED ACTIVATION OF ENDOGENOUS GENES” which is acontinuation-in-part of U.S. application Ser. No. 09/263,814 of John J.Harrington, Bruce Sherf, and Stephen Rundlett, entitled “Compositionsand Methods for Non-targeted Activation of Endogenous Genes,” filed Mar.8, 1999, now abandoned, which is a continuation-in-part of U.S.application Ser. No. 09/253,022, filed Feb. 19, 1999, now abandoned,which is a continuation-in-part of U.S. application Ser. No. 09/159,643,filed Sep. 24, 1998, now abandoned, which is a continuation-in-part ofU.S. application Ser. No. 08/941,223, filed Sep. 26, 1997, nowabandoned, the disclosures of all of which are incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is in the fields of molecular biology and cellularbiology. The invention is directed generally to activation of geneexpression or causing over-expression of a gene by recombination methodsin situ. More specifically, the invention is directed to activation ofendogenous genes by non-targeted integration of specialized activationvectors, which are provided by the invention, into the genome of a hostcell. The invention also is directed to methods for the identification,activation, and isolation of genes that were heretofore undiscoverable,and to host cells and vectors comprising such isolated genes. Theinvention also is directed to isolated genes, gene products, nucleicacid molecules, and compositions comprising such genes, gene productsand nucleic acid molecules, that may be used in a variety of therapeuticand diagnostic applications. Thus, by the present invention, endogenousgenes, including those associated with human disease and development,may be identified, activated, and isolated without prior knowledge ofthe sequence, structure, function, or expression profile of the genes.

2. Related Art

Identification and over-expression of novel genes associated with humandisease is an important step towards developing new therapeutic drugs.Current approaches to creating libraries of cells for proteinover-expression are based on the production and cloning of cDNA. Thus,in order to identify a new gene using this approach, the gene must beexpressed in the cells that were used to make the library. The gene alsomust be expressed at sufficient levels to be adequately represented inthe library. This is problematic because many genes are expressed onlyin very low quantities, in a rare population of cells, or during shortdevelopmental periods.

Furthermore, because of the large size of some mRNAs, it is difficult orimpossible to produce fill length cDNA molecules capable of expressingthe biologically active protein. Lack of full-length cDNA molecules hasalso been observed for small mRNAs and is thought to be related tosequences in the message that are difficult to produce by reversetranscription or that are unstable during propagation in bacteria. As aresult, even the most complete cDNA libraries express only a fraction ofthe entire set of possible genes.

Finally, many cDNA libraries are produced in bacterial vectors. Use ofthese vectors to express biologically active mammalian proteins isseverely limited since most mammalian proteins do not fold correctlyand/or are improperly glycosylated in bacteria.

Therefore, a method for creating a more representative library forprotein expression, capable of facilitating faithful expression ofbiologically active proteins, would be extremely valuable.

Current methods for over-expressing proteins involve cloning the gene ofinterest and placing it, in a construct, next to a suitablepromoter/enhancer, polyadenylation signal, and splice site, andintroducing the construct into an appropriate host cell.

An alternative approach involves the use of homologous recombination toactivate gene expression by targeting a strong promoter or otherregulatory sequence to a previously identified gene.

WO 90/14092 describes in situ modification of genes, in mammalian cells,encoding proteins of interest. This application describessingle-stranded oligonucleotides for site-directed modification of genesencoding proteins of interest. A marker may also be included. However,the methods are limited to providing an oligonucleotide sequencesubstantially homologous to a target site. Thus, the method requiresknowledge of the site required for activation by site-directedmodification and homologous recombination. Novel genes are notdiscoverable by such methods.

WO 91/06667 describes methods for expressing a mammalian gene in situ.With this method, an amplifiable gene is introduced next to a targetgene by homologous recombination. When the cell is then grown in theappropriate medium, both the amplifiable gene and the target gene areamplified and there is enhanced expression of the target gene. As above,methods of introducing the amplifiable gene are limited to homologousrecombination, and are not useful for activating novel genes whosesequence (or existence) is unknown.

WO 91/01140 describes the inactivation of endogenous genes bymodification of cells by homologous recombination. By these methods,homologous recombination is used to modify and inactivate genes and toproduce cells which can serve as donors in gene therapy.

WO 92/20808 describes methods for modifying genomic target sites insitu. The modifications are described as being small, for example,changing single bases in DNA. The method relies upon genomicmodification using homologous DNA for targeting.

WO 92/19255 describes a method for enhancing the expression of a targetgene, achieved by homologous recombination in which a DNA sequence isintegrated into the genome or large genomic fragment. This modifiedsequence can then be transferred to a secondary host for expression. Anamplifiable gene can be integrated next to the target gene so that thetarget region can be amplified for enhanced expression. Homologousrecombination is necessary to this targeted approach.

WO 93/09222 describes methods of making proteins by activating anendogenous gene encoding a desired product. A regulatory region istargeted by homologous recombination and replacing or disabling theregion normally associated with the gene whose expression is desired.This disabling or replacement causes the gene to be expressed at levelshigher than normal.

WO 94/12650 describes a method for activating expression of andamplifying an endogenous gene in situ in a cell, which gene is notexpressed or is not expressed at desired levels in the cell. The cell istransfected with exogenous DNA sequences which repair, alter, delete, orreplace a sequence present in the cell or which are regulatory sequencesnot normally functionally linked to the endogenous gene in the cell. Inorder to do this, DNA sequences homologous to genomic DNA sequences at apreselected site are used to target the endogenous gene. In addition,amplifiable DNA encoding a selectable marker can be included. Byculturing the homologously recombinant cells under conditions thatselect for amplification, both the endogenous gene and the amplifiablemarker are co-amplified and expression of the gene increased.

WO 95/31560 describes DNA constructs for homologous recombination. Theconstructs include a targeting sequence, a regulatory sequence, an exon,and an unpaired splice donor site. The targeting is achieved byhomologous recombination of the construct with genomic sequences in thecell and allows the production of a protein in vitro or in vivo.

WO 96/29411 describes methods using an exogenous regulatory sequence, anexogenous exon, either coding or non-coding, and a splice donor siteintroduced into a preselected site in the genome by homologousrecombination. In this application, the introduced DNA is positioned sothat the transcripts under control of the exogenous regulatory regioninclude both the exogenous exon and endogenous exons present in eitherthe thrombopoietin, DNase I, or β-interferon genes, resulting intranscripts in which the exogenous and exogenous exons are operablylinked. The novel transcription units are produced by homologousrecombination.

U.S. Pat. No. 5,272,071 describes the transcriptional activation oftranscriptionally silent genes in a cell by inserting a DNA regulatoryelement capable of promoting the expression of a gene normally expressedin that cell. The regulatory element is inserted so that it is operablylinked to the normally silent gene. The insertion is accomplished bymeans of homologous recombination by creating a DNA construct with asegment of the normally silent gene (the target DNA) and the DNAregulatory element used to induce the desired transcription.

U.S. Pat. No. 5,578,461 discusses activating expression of mammaliantarget genes by homologous recombination. A DNA sequence is integratedinto the genome or a large genomic fragment to enhance the expression ofthe target gene. The modified construct can then be transferred to asecondary host. An amplifiable gene can be integrated adjacent to thetarget gene so that the target region is amplified for enhancedexpression.

Both of the above approaches (construction of an over-expressingconstruct by cloning or by homologous recombination in vivo) require thegene to be cloned and sequenced before it can be over-expressed.Furthermore, using homologous recombination, the genomic sequence andstructure must also be known.

Unfortunately, many genes have not yet been identified and/or sequenced.Thus, a method for over-expressing a gene of interest, whether or not ithas been previously cloned, and whether or not its sequence andstructure are known, would be useful.

BRIEF SUMMARY OF THE INVENTION

The invention is, therefore, generally directed to methods forover-expressing an endogenous gene in a cell, comprising introducing avector containing a transcriptional regulatory sequence into the cell,allowing the vector to integrate into the genome of the cell bynon-homologous recombination, and allowing over-expression of theendogenous gene in the cell. The method does not require previousknowledge of the sequence of the endogenous gene or even of theexistence of the gene. Hence, the invention is directed to non-targetedgene activation, which as used herein means the activation of endogenousgenes by non-targeted or non-homologous (as opposed to targeted orhomologous) integration of specialized activation vectors into thegenome of a host cell.

The invention also encompasses novel vector constructs for activatinggene expression or over-expressing a gene through non-homologousrecombination. The novel construct lacks homologous targeting sequences.That is, it does not contain nucleotide sequences that target host cellDNA and promote homologous recombination at the target site, causingover-expressing of a cellular gene via the introduced transcriptionalregulatory sequence.

Novel vector constructs include a vector containing a transcriptionalregulatory sequence operably linked to an unpaired splice donor sequenceand further contains one or more amplifiable markers.

Novel vector constructs include constructs with a transcriptionalregulatory sequence operably linked to a translational start codon, asignal secretion sequence, and an unpaired splice donor site; constructswith a transcriptional regulatory sequence, operably linked to atranslation start codon, an epitope tag, and an unpaired splice donorsite; constructs containing a transcriptional regulatory sequenceoperably linked to a translational start codon, a signal sequence and anepitope tag, and an unpaired splice donor site; constructs containing atranscriptional regulatory sequence operably linked to a translationstart codon, a signal secretion sequence, an epitope tag, and asequence-specific protease site, and an unpaired splice donor site.

The vector construct can contain one or more selectable markers forrecombinant host cell selection. Alternatively, selection can beeffected by phenotypic selection for a trait provided by the activatedendogenous gene product.

These vectors, and indeed any of the vectors disclosed herein, andvariants of the vectors that will be readily recognized by one ofordinary skill in the art, can be used in any of the methods describedherein to form any of the compositions producible by these methods.

The transcriptional regulatory sequence used in the vector constructs ofthe invention includes, but is not limited to, a promoter. In preferredembodiments, the promoter is a viral promoter. In highly preferredembodiments, the viral promoter is the cytomegalovirus immediate earlypromoter. In alternative embodiments, the promoter is a cellular,non-viral promoter or inducible promoter.

The transcriptional regulatory sequence used in the vector construct ofthe invention may also include, but is not limited to, an enhancer. Inpreferred embodiments, the enhancer is a viral enhancer. In highlypreferred embodiments, the viral enhancer is the cytomegalovirusimmediate early enhancer. In alternative embodiments, the enhancer is acellular non-viral enhancer.

In preferred embodiments of the methods described herein, the vectorconstruct be, or may contain, linear RNA or DNA.

The cell containing the vector may be screened for expression of thegene.

The cell over-expressing the gene can be cultured in vitro underconditions favoring the production, by the cell, of desired amounts ofthe gene product (also referred to interchangeably herein as the“expression product”) of the endogenous gene that has been activated orwhose expression has been increased. The expression product can then beisolated and purified to use, for example, in protein therapy or drugdiscovery

Alternatively, the cell expressing the desired gene product can beallowed to express the gene product in vivo. In certain such aspects ofthe invention, the cell containing a vector construct of the inventionintegrated into its genome may be introduced into a eukaryote (such as avertebrate, particularly a mammal, more particularly a human) underconditions favoring the over expression or activation of the gene by thecell in vivo in the eukaryote. In related such aspects of the invention,the cell may be isolated and cloned prior to being introduced into theeukaryote.

The invention is also directed to methods for over-expressing anendogenous gene in a cell, comprising introducing a vector containing atranscriptional regulatory sequence and one or more amplifiable markersinto the cell, allowing the vector to integrate into the genome of thecell by non-homologous recombination, and allowing over-expression ofthe endogenous gene in the cell.

The cell containing the vector may be screened for over-expression ofthe gene.

The cell over-expressing the gene is cultured such that amplification ofthe endogenous gene is obtained. The cell can then be cultured in vitroso as to produce desired amounts of the gene product of the amplifiedendogenous gene that has been activated or whose expression has beenincreased. The gene product can then be isolated and purified.

Alternatively, following amplification, the cell can be allowed toexpress the endogenous gene and produce desired amounts of the geneproduct in vivo.

It is to be understood, however, that any vector used in the methodsdescribed herein can include one or more amplifiable markers. Thereby,amplification of both the vector and the DNA of interest (i.e.,containing the over-expressed gene) occurs in the cell, and furtherenhanced expression of the endogenous gene is obtained. Accordingly,methods can include a step in which the endogenous gene is amplified.

The invention is also directed to methods for over-expressing anendogenous gene in a cell comprising introducing a vector containing atranscriptional regulatory sequence and one or more amplifiable markersinto the cell, allowing the vector to integrate into the genome of thecell by non-homologous recombination, and allowing over-expression ofthe endogenous gene in the cell.

The cell containing the vector may be screened for expression of thegene.

The cell over-expressing the gene can be cultured in vitro so as toproduce desirable amounts of the gene product of the endogenous genewhose expression has been activated or increased. The gene product canthen be isolated and purified.

Alternatively, the cell can be allowed to express the desired geneproduct in vivo.

The vector construct can consist essentially of the transcriptionalregulatory sequence.

The vector construct can consist essentially of the transcriptionalregulatory sequence and one or more amplifiable markers.

The vector construct can consist essentially of the transcriptionalregulatory sequence and the splice donor sequence.

Any of the vector constructs of the invention can also include asecretion signal sequence. The secretion signal sequence is arranged inthe construct so that it will be operably linked to the activatedendogenous protein. Thereby, secretion of the protein of interest occursin the cell, and purification of that protein is facilitated.Accordingly, methods can include a step in which the protein expressionproduct is secreted from the cell.

The invention also encompasses cells made by any of the above methods.The invention encompasses cells containing the vector constructs, cellsin which the vector constructs have integrated into the cellular genome,and cells which are over-expressing desired gene products from anendogenous gene, over-expression being driven by the introducedtranscriptional regulatory sequence.

The cells can be isolated and cloned.

The methods can be carried out in any cell of eukaryotic origin, such asfungal, plant or animal. In preferred embodiments, the methods of theinvention may be carried out in vertebrate cells, and particularlymammalian cells including but not limited to rat, mouse, bovine,porcine, sheep, goat and human cells, and more particularly in humancells.

A single cell made by the methods described above can over-express asingle gene or more than one gene. More than one gene in a cell can beactivated by the integration of a single type of construct into multiplelocations in the genome. Similarly, more than one gene in a cell can beactivated by the integration of multiple constructs (i.e., more than onetype of construct) into multiple locations in the genome. Therefore, acell can contain only one type of vector construct or different types ofconstructs, each capable of activating an endogenous gene.

The invention is also directed to methods for making the cells describedabove by one or more of the following: introducing one or more of thevector constructs of the invention into a cell; allowing the introducedconstruct(s) to integrate into the genome of the cell by non-homologousrecombination; allowing over-expression of one or more endogenous genesin the cell; and isolating and cloning the cell. The invention is alsodirected to cells produced by such methods, which may be isolated cells.

The invention also encompasses methods for using the cells describedabove to over-express a gene, such as an endogenous cellular gene, thathas been characterized (for example, sequenced), uncharacterized (forexample, a gene whose function is known but which has not been cloned orsequenced), or a gene whose existence was, prior to over-expression,unknown. The cells can be used to produce desired amounts of anexpression product in vitro or in vivo. If desired, this expressionproduct can then be isolated and purified, for example by cell lysis orby isolation from the growth medium (as when the vector contains asecretion signal sequence).

The invention also encompasses libraries of cells made by the abovedescribed methods. A library can encompass all of the clones from asingle transfection experiment or a subset of clones from a singletransfection experiment. The subset can over-express the same gene ormore than one gene, for example, a class of genes. The transfection canhave been done with a single construct or with more than one construct.

A library can also be formed by combining all of the recombinant cellsfrom two or more transfection experiments, by combining one or moresubsets of cells from a single transfection experiment or by combiningsubsets of cells from separate transfection experiments. The resultinglibrary can express the same gene, or more than one gene, for example, aclass of genes. Again, in each of these individual transfections, aunique construct or more than one construct can be used.

Libraries can be formed from the same cell type or different cell types.

The invention is also directed to methods for making libraries byselecting various subsets of cells from the same or differenttransfection experiments.

The invention is also directed to methods of using the above-describedcells or libraries of cells to over-express or activate endogenousgenes, or to obtain the gene expression products of such over-expressedor activated genes. According to this aspect of the invention, the cellor library may be screened for the expression of the gene and cells thatexpress the desired gene product may be selected. The cell can then beused to isolate or purify the gene product for subsequent use.Expression in the cell can occur by culturing the cell in vitro, underconditions favoring the production of the expression product of theendogenous gene by the cell, or by allowing the cell to express the genein vivo.

In preferred embodiments of the invention, the methods include a processwherein the expression product is isolated or purified. In highlypreferred embodiments, the cells expressing the endogenous gene productare cultured under conditions favoring production of sufficient amountsof gene product for commercial application, and especially fordiagnostic, therapeutic and drug discovery uses.

Any of the methods can further comprise introducing double-strand breaksinto the genomic DNA in the cell prior to or simultaneously with vectorintegration.

The invention also is directed to vector constructs that are useful foractivating expression of endogenous genes and for isolating the mRNA andcDNA corresponding to the activated genes.

In one such embodiment, the vector construct may comprise (a) a firsttranscriptional regulatory sequence operably linked to a first unpairedsplice donor sequence; (b) a second transcriptional regulatory sequenceoperably linked to a second unpaired splice donor sequence; and (c) alinearization site, which may be located between the first and secondtranscriptional regulatory sequences. According to the invention, whenthe vector construct is transformed into a host cell and then integratesinto the genome of the host cell, the first transcriptional regulatorysequence is preferably in an inverted orientation relative to theorientation of the second transcriptional regulatory sequence. Incertain preferred such embodiments, the vector may be rendered linear bycleavage at the linearization site.

In another embodiment, the invention provides a linear vector constructhaving a 3′ end and a 5′ end, comprising a transcriptional regulatorysequence operably linked to an unpaired spliced donor site, wherein thetranscriptional regulatory sequence is oriented in the linear vectorconstruct in an orientation that directs transcription towards the 3′end or the 5′ end of the linear vector construct.

In another embodiment, the invention provides a vector constructcomprising, in sequential order, (a) a transcriptional regulatorysequence, (b) an unpaired splice donor site, (c) a rare cuttingrestriction site, and (d) a linearization site.

In another embodiment, the invention provides a vector constructcomprising (a) a first transcriptional regulatory sequence operablylinked to a selectable marker lacking a polyadenylation signal; and (b)a second transcriptional regulatory sequence operably linked to anexon-splice donor site complex, wherein the first transcriptionalregulatory sequence is in the same orientation in the vector constructas is the second transcriptional regulatory sequence, and wherein thefirst transcriptional regulatory sequence is upstream of the secondtranscriptional regulatory sequence in the vector construct.

In additional embodiments, the invention provides vector constructscomprising a transcriptional regulatory sequence operably linked to aselectable marker lacking a polyadenylation signal, and furthercomprising an unpaired splice donor site.

In another embodiment, the invention provides vector constructscomprising a first transcriptional regulatory sequence operably linkedto a selectable marker lacking a polyadenylation signal, and furthercomprising a second transcriptional regulatory sequence operably linkedto an unpaired splice donor site.

According to the invention, the transcriptional regulatory sequence (orfirst or second transcriptional regulatory sequence, in vectorconstructs having more than one transcriptional regulatory sequence) maybe a promoter, an enhancer, or a repressor, and is preferably apromoter, including an animal cell promoter, a plant cell promoter, or afungal cell promoter, most preferably a promoter selected from the groupconsisting of a CMV immediate early gene promoter, an SV40 T antigenpromoter, and a β-actin promoter. Other promoters of animal, plant, orfungal cell origin that may be used in accordance with the invention areknown in the art and will be familiar to one of ordinary skill in viewof the teachings herein.

The selectable marker used in the vector constructs of the invention maybe any marker or marker gene that, upon integration of a vectorcontaining the selectable marker into the host cell genome, permits theselection of a cell containing or expressing the marker gene. Suitablesuch selectable markers include, but are not limited to, a neomycingene, a hypoxanthine phosphribosyl transferase gene, a puromycin gene, adihydrooratase gene, a glutamine synthetase gene, a histidine D gene, acarbamyl phosphate synthase gene, a dihydrofolate reductase gene, amultidrug resistance 1 gene, an aspartate transcarbamylase gene,axanthine-guanine phosphoribosyl transferase gene, an adenosinedeaminase gene, and a thymidine kinase gene.

In related embodiments, the invention provides vector constructscomprising a positive selectable marker, a negative selectable marker,and an unpaired splice donor site, wherein the positive and negativeselectable markers and the splice donor site are oriented in the vectorconstruct in an orientation that results in expression of the positiveselectable marker in active form, and either non-expression of saidnegative selectable marker or expression of the negative selectablemarker in inactive form, when the vector construct is integrated intothe genome of a eukaryotic host cell and activates an endogenous gene inthe genome. In certain preferred such embodiments, either the positiveselection marker, the negative selection marker, or both, may lack apolyadenylation signal. The positive selection marker used in theseaspects of the invention may be any selection marker that, uponexpression, produces a protein capable of facilitating the isolation ofcells expressing the marker, including but not limited to a neomycingene, a hypoxanthine phosphribosyl transferase gene, a puromycin gene, adihydrooratase gene, a glutamine synthetase gene, a histidine D gene, acarbamyl phosphate synthase gene, a dihydrofolate reductase gene, amultidrug resistance 1 gene, an aspartate transcarbamylase gene, axanthine-guanine phosphoribosyl transferase gene, or an adenosinedeaminase gene. Analogously, the negative selection marker used in theseaspects of the invention may be any selection marker that, uponexpression, produces a protein capable of facilitating removal of cellsexpressing the marker, including but not limited to a hypoxanthinephosphribosyl transferase gene, a thymidine kinase gene, or a diphtheriatoxin gene.

The invention also is directed to eukaryotic host cells, which may beisolated host cells, comprising one or more of the vector constructs ofthe invention. Preferred such eukaryotic host cells include, but are notlimited to, animal cells (including, but not limited to, mammalian(particularly human) cells, insect cells, avian cells, annelid cells,amphibian cells, reptilian cells, and fish cells), plant cells, andfungal (particularly yeast) cells. In certain such host cells, thevector construct may be integrated into the genome of the host cell.

The invention also is directed to primer molecules comprising aPCR-amplifiable sequence and a degenerate 3′ terminus. Primer moleculesaccording to this aspect of the invention preferably have the generalstructure:

5′-(dT)_(a)-X-N_(b)-TTTATT-3′,

wherein a is a whole number from 1 to 100 (preferably from 10 to 30), Xis a PCR-amplifiable sequence consisting of a nucleic acid sequence ofabout 10-20 nucleotides in length, N is any nucleotide, and b is a wholenumber from 0 to 6. One preferred such primer has the nucleotidesequence 5′-TTTTTTTTTTTTCGTCAGCGGCCGCATCNNNNTTTATT-3′ (SEQ ID NO:10). Inrelated embodiments, the primer molecules according to this aspect ofthe invention may be biotinylated.

The invention also is directed to methods for first strand cDNAsynthesis comprising (a) annealing a first primer of the invention (suchas the primer described above) to an RNA template molecule to form anfirst primer-RNA complex, and (b) treating this first primer-RNA complexwith reverse transcriptase and one or more deoxynucleotide triphosphatemolecules under conditions favoring the reverse transcription of thefirst primer-RNA complex to synthesize a first strand cDNA.

The invention also is directed to methods for isolating activated genes,particularly from a host cell genome. These methods of the inventionexploit the structure of the mRNA molecules produced using thenon-targeted gene activation vectors of the invention. One such methodof the invention comprises, for example, (a) introducing a vectorconstruct comprising a transcriptional regulatory sequence and anunpaired splice donor site into a host cell (preferably one of theeukaryotic host cells described above), (b) allowing the vectorconstruct to integrate into the genome of the host cell bynon-homologous recombination, under conditions such that the vectoractivates an endogenous gene comprising an exon in the genome, (c)isolating RNA from the host cell, (d) synthesizing first strand cDNAaccording to the method of the invention described above, (e) annealinga second primer specific for the vector-encoded exon to the first strandcDNA to create a second primer-first strand cDNA complex, and (f)contacting the second primer-first strand cDNA complex with a DNApolymerase under conditions favoring the production of a second strandcDNA substantially complementary to the first strand cDNA Methodsaccording to this aspect of the invention may comprise one or moreadditional steps, such as treating the second strand cDNA with arestriction enzyme that cleaves at a restriction site located on thevector downstream of the unpaired splice donor site, or amplifying thesecond strand cDNA using a third primer specific for the vector-encodedexon and a fourth primer specific for the second primer. The inventionalso is directed to isolated genes, produced according to these methods,and to vectors (which may be expression vectors) and host cellscomprising these isolated genes. The invention also is directed tomethods of producing a polypeptide, comprising cultivating a host cellcomprising the isolated gene (or a vector, particularly an expressionvector, comprising the isolated gene), and culturing the host cell underconditions favoring the expression by the host cell of a polypeptideencoded by the isolated gene. The invention also provides additionalmethods of producing a polypeptide, comprising introducing into a hostcell a vector comprising a transcriptional regulatory sequence operablylinked to an exonic region followed by an unpaired splice donor site,and culturing the host cell under conditions favoring the expression bysaid host cell of a polypeptide encoded by the exonic region, whereinthe exon contains a translational start site positioned at any of theopen reading frame positions relative to the 5′-most base of theunpaired splice donor site (e.g., the “A” in the ATG start codon may beat position −3 or at an increment of 3 bases upstream therefrom (e.g.,−6, −9, −12, −15, −18, etc.), at position −2 or at an increment of 3bases upstream therefrom (e.g., −5, −8, −11, −14, −17, −20, etc.), or atposition −1 or at an increment of 3 bases upstream therefrom (e.g., −4,−7, −10, −13, −16, −19, etc.), relative to the 5′-most base of thesplice donor site). In related embodiments, the methods of the inventionmay further comprise isolating the polypeptide. The invention also isdirected to polypeptides, which may or may not be isolated polypeptides,produced according to these methods.

Other preferred embodiments of the present invention will be apparent toone of ordinary skill in light of the following drawings and descriptionof the invention, and of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of gene activation events described herein.The activation construct is transfected into cells and allowed tointegrate into the host cell chromosomes at DNA breaks. If breakageoccurs upstream of a gene of interest (e.g., Epo), and the appropriateactivation construct integrates at the break such that its regulatorysequence becomes operably linked to the gene of interest, activation ofthe gene will occur. Transcription and splicing produce a chimeric RNAmolecule containing exonic sequences from the activation construct andfrom the endogenous gene. Subsequent translation will result in theproduction of the protein of interest. Following isolation of therecombinant cell, gene expression can be further enhanced via geneamplification.

FIG. 2. Schematic diagram of non-translated activation constructs. Thearrows denote promoter sequences. The exonic sequences are shown as openboxes and the splice donor sequence is indicated by S/D. Constructnumbers corresponding to the description below are shown on the left.The selectable and amplifiable markers are not shown.

FIG. 3. Schematic diagram of translated activation constructs. Thearrows denote promoter sequences. The exonic sequences are shown as openboxes and the splice donor sequence is indicated by S/D. The translated,signal peptide, epitope tag, and protease cleavage sequences are shownin the legend below the constructs. Construct numbers corresponding tothe description below are shown on the left. The selectable andamplifiable markers are not shown.

FIG. 4. Schematic diagram of an activation construct capable ofactivating endogenous genes.

FIGS. 5A-5D. Nucleotide sequence of pRIG8R1-CD2 (SEQ ID NO:7).

FIGS. 6A-6C. Nucleotide sequence of pRIG8R2-CD2 (SEQ ID NO:8).

FIGS. 7A-7C. Nucleotide sequence of pRIG8R3-CD2 (SEQ ID NO:9).

FIGS. 8A-8F. Examples of poly(A) trap vectors. Each vector isillustrated schematically in its linearized form. Each horizontal linerepresents a DNA molecule. The arrows denote promoter sequences locatedon the DNA molecule, and face in the direction of transcription.Transcribed regions include all sequences located downstream of apromoter. Untranslated regions are designated by hatched boxes and openreading frames are designated by open boxes. The following designationswere used: splice donor site (S/D), signal secretion sequence (SP),epitope tag (ET), neomycin resistance gene (Neo). In the vectorsdepicted in FIGS. 8B-8E, it is possible to omit the splice donor siteimmediately downstream of the Neo gene. In vectors lacking a splicedonor site between the neo gene and the downstream promoter, the Neotranscript will utilize the splice donor site located 3′ of thedownstream promoter. In addition, as shown in the vectors depicted inFIGS. 8B-8E, a downstream promoter may drive expression of an exon. Itis recognized that this exon, when present, may encode codons in anyreading frame. Using multiple vectors, codons in each of the 3 possiblereading frames can be created.

FIGS. 9A-9F. Examples of splice acceptor trap vectors containing apositive and a negative selectable marker driven from a single promoter.Each vector is illustrated schematically in its linearized form. Eachhorizontal line represents a DNA molecule. The arrows denote promotersequences located on the DNA molecule, and face in the direction oftranscription. Transcribed regions include all sequences locateddownstream of a promoter. Untranslated regions are designated by hatchedboxes. Poly(A) signals are not present in these examples. As describedin the specification, however, poly(A) signals may be placed on thevector 3′ of either or both selectable markers. The followingdesignations were used: splice donor site (S/D), signal secretionsequence (SP), epitope tag (ET), internal ribosome entry site (ires),hypoxanthine phosphoribosyl transferase (HPRT), and neomycin resistancegene (Neo). In these examples, Neo represents the positive selectablemarker and HPRT represents the negative selectable marker. In thevectors shown in FIGS. 9C and 9F, the region designated exon contains atranslation start codon. As described in the Detailed Description, theexon may encode a methionine residue, a partial signal sequence, a fullsignal secretion sequence, a portion of a protein, or an epitope tag. Inaddition, the codons may be present in any reading frame relative to thesplice donor site. In other vector examples not shown, the regiondesignated exon lacks a translation start codon.

FIGS. 10A-10F. Examples of splice acceptor trap vectors containing apositive and negative selectable marker driven from different promoters.Each vector is illustrated schematically in its linearized form. Eachhorizontal line represents a DNA molecule. The arrows denote promotersequences located on the DNA molecule, and face in the direction oftranscription. Transcribed regions include all sequences locateddownstream of a promoter. Untranslated regions are designated by hatchedboxes. Poly(A) signals are not present in these examples. As describedin the specification, however, poly(A) signals may be placed on thevector 3′ of either or both selectable markers. The followingdesignations were used: splice donor site (S/D), internal ribosome entrysite (ires), hypoxanthine phosphoribosyl transferase (HPRT), andneomycin resistance gene (Neo). In the vectors shown in FIGS. 10A-10F,Neo represents the positive selectable marker and HPRT represents thenegative selectable marker. As shown, the vectors depicted in FIGS.10A-10F do not contain a splice donor site 3′ of the Neo gene; however,in other vectors not shown, a splice donor site may be located 3′ of theNeo gene to facilitate splicing of the positive selection marker to anendogenous exon In the vectors shown in FIGS. 10C and 10F, the regiondesignated exon contains a translation start codon. As described in theDetailed Description, the exon may encode a methionine residue, apartial signal sequence, a full signal secretion sequence, a portion ofa protein, or an epitope tag. In addition, the codons may be present inany reading frame relative to the splice donor site. In other vectorexamples not shown, the region designated exon lacks a translation startcodon.

FIGS. 11A-11C. Schematic diagram of bidirectional activation vectors.The arrows denote promoter sequences. The exons are shown as checkeredboxes and splice donor sites are indicated by S/D. The hatched boxesindicate exon sequences operably linked to the upstream promoter. It isunderstood that the exons on these vectors may be untranslated, or maycontain a start codon and additional codons as described herein. Asillustrated in the vectors depicted in FIGS. 11B-11C, the vectors maycontain a selectable marker. In these vectors, the neomycin resistance(Neo) gene is illustrated. In FIG. 11B, a polyadenylation signal (pA) islocated downstream of the selectable marker. In FIG. 11C,polyadenylation signals are omitted from the vector.

FIGS. 12A-12G. Examples of vectors useful for recovering exon I fromactivated endogenous genes. Each vector is illustrated schematically inits linearized form. Each horizontal line represents a DNA molecule. Thearrows denote promoter sequences located on the DNA molecule, and facein the direction of transcription. Transcribed regions include allsequences located downstream of a promoter. Untranslated regions aredesignated by hatched boxes. Poly(A) signals are not present in thevectors depicted. As discussed in the Detailed Description, however,poly(A) signals may be placed on the vector 3′ of either or bothselectable markers. The following designations were used: splice donorsite (S/D), internal ribosome entry site (ires), hypoxanthinephosphoribosyl transferase (HPRT), and neomycin resistance gene (Neo).In these examples, Neo represents the positive selectable marker andHPRT represents the negative selectable marker. It is also recognizedthat in these examples, the region designated exon, when present, lacksa translation start codon. In other examples not shown, the regiondesignated exon contains a translation start codon. Furthermore, whenthe vector exon contains a translation start codon, the exon may encodea methionine residue, a partial signal sequence, a fill signal secretionsequence, a portion of a protein, or an epitope tag. In addition, thecodons may be present in each reading frame relative to the splice donorsite.

FIG. 13. Illustration depicting two transcripts produced from theintegrated vectors described in FIGS. 12A-12G. DNA strands are depictedas horizontal lines. Vector DNA is shown as a black line. Endogenousgenomic DNA is shown as a grey line. Rectangles depict exons.Vector-encoded exons are shown as open rectangles, while endogenousexons are shown as shaded boxes. S/D denotes a splice donor site.Following integration, the vector encoded promoters activatetranscription of the endogenous gene. Transcription resulting from theupstream promoter produces a spliced RNA molecule containing the vectorencoded exon joined to the second and subsequent exons from anendogenous gene. Transcription from the downstream promoter, on theother hand, produces a transcript containing the sequences downstream ofthe integrated joined to exon I and the subsequent exons from anendogenous gene.

FIGS. 14A-14B Nucleotide sequence of pRIG1 (SEQ ID NO:18).

FIGS. 15A-15B. Nucleotide sequence of pRIG21b (SEQ ID NO:19).

FIGS. 16A-16B. Nucleotide sequence of pRIG22b (SEQ ID NO:20).

FIGS. 17A-17G. Examples of poly(A) trap vectors. Each vector isillustrated schematically in its linearized form. Each horizontal linerepresents a DNA molecule. The arrows denote promoter sequences locatedon the DNA molecule, and face in the direction of transcription.Transcribed regions include all sequences located downstream of apromoter. Boxes indicate exons. Hatched boxes indicate untranslatedregions. The following designations were used: splice donor site (S/D),signal secretion sequence (SP), epitope tag (ET), neomycin resistancegene (Neo), vector promoter #1 (VP#1), and vector promoter #2 (VP#2). Asshown in the vectors depicted in FIGS. 17C-17G, a promoter operablylinked to an exon and an unpaired splice donor site can be positionedupstream of the selectable marker. It is recognized that this exon, whenpresent, may encode codons a start codon in any reading frame relativeto the splice donor site. To activate protein expression from genes withdifferent reading frames, three separate vectors can be used, each witha start codon in a different reading frame relative to the splice donorsite.

FIG. 18. Illustration of the transcripts produced by the vector fromFIG. 17C upon integration into a host cell genome upstream of amulti-exon endogenous gene. Each horizontal line represents a DNAmolecule. Vertical lines running through the DNA strand mark theupstream and downstream vector/cellular genome boundaries. The arrowsdenote promoter sequences located on the DNA molecule, and face in thedirection of transcription. Transcribed regions include all sequenceslocated downstream of a promoter. Boxes indicate exons. Hatched boxesindicate untranslated regions. The endogenous exons are numbered usingroman numerals. The following designations were used: splice donor site(S/D), neomycin resistance gene (Neo), vector promoter #1 (VP#1), vectorpromoter #2 (VP#2), endogenous promoter (EP) and polyadenylation signal(pA). Following integration, vector promoter #1 expresses a chimerictranscript containing the Neo gene linked to the genomic sequencesdownstream of the integration site, including the processed (spliced)exons from the endogenous gene. Since transcript #1 contains a poly (A)signal from the endogenous gene, the Neo gene product will beefficiently produced, thereby conferring drug resistance on the cell. Inaddition to transcript #1, the integrated vector will generate a secondtranscript, designated transcript #2, originating from vectorpromoter#2. The structure of transcript #2 facilitates efficienttranslation of the protein encoded by the endogenous gene. Asexemplified in FIG. 17, vectors containing alternative codinginformation in the vector encoded exon can be used to produce differentchimeric proteins, containing, for example, signal sequences and/orepitope tags.

FIG. 19. Example of dual positive selectable marker vector. The vectoris illustrated schematically in its linearized form. The horizontal linerepresents a DNA molecule. The arrows denote promoter sequences locatedon the DNA molecule, and face in the direction of transcription.Transcribed regions include all sequences located downstream of apromoter. Boxes indicate exons. Hatched boxes indicate untranslatedregions. Poly(A) signals are not present in these examples. Thefollowing designations were used: splice donor site (S/D), hygromycinresistance gene (Hyg), neomycin resistance gene (Neo), vector promoter#1, and vector promoter #2.

FIGS. 20A-20B. Examples of transcripts produced by a dual positiveselectable marker vector integrated into a host cell genome adjacent toan endogenous gene. FIG. 20A illustrates the transcripts produced uponvector integration near a multi-exon gene. FIG. 20B illustrates thetranscripts produced upon vector integration near a single exon gene.Each horizontal line represents a DNA molecule. Vertical lines runningthrough the DNA strand mark the upstream and downstream vector/cellulargenome boundaries. The arrows denote promoter sequences located on theDNA molecule, and face in the direction of transcription. Transcribedregions include all sequences located downstream of each promoter Boxesindicate exons. Hatched boxes indicate untranslated regions. Theendogenous exons are numbered using roman numerals. The followingdesignations were used: splice donor site (S/D), hygromycin resistancegene (Hyg), neomycin resistance gene (Neo), vector promoter #1 (VP#1),vector promoter #2 (VP#2), endogenous promoter (EP), and polyadenylationsignal (pA). Following integration, vector promoter #1 expresses achimeric transcript containing the Hyg gene linked to the genomicsequences downstream of the integration site, including the processed(spliced) exons from the endogenous gene. Since transcript #1 contains apoly (A) signal from the endogenous gene, the Hyg gene product will beefficiently produced, thereby conferring drug resistance on the cell. Inaddition to transcript #1, the integrated vector will generate a secondtranscript, designated transcript #2, originating from vectorpromoter#2. In FIG. 20A, the neo gene is removed from transcript #2 uponsplicing from the vector encoded splice donor site, and the firstendogenous splice acceptor located downstream of the vector integrationsite (i.e. exon II in this example). Since multi-exon genes containsplice acceptor sites at the 5′ end of each exon (except exon I), theneo gene will be removed from transcript #2 in cells in which the vectorhas integrated near, and transcriptionally activated, a multi-exon gene.As a result, cells having activated multi-exon genes may be eliminatedby selecting with G418 and hygromycin. In FIG. 20B, the neo gene is notremoved from transcript #2 by splicing, since single exon genes do notcontain any splice acceptor sequences. Thus, cells containing a vectorintegrated near single exon genes will survive double selection withG418 and hygromycin. These cells can be used to efficiently isolate theactivated single exon genes using methods described herein.

FIG. 21 Examples of dual trap vectors containing a positive and anegative selectable marker. Each vector is illustrated schematically inits linearized form. Each horizontal line represents a DNA molecule. Thearrows denote promoter sequences located on the DNA molecule, and facein the direction of transcription. Transcribed regions include allsequences located downstream of a promoter. Boxes indicate exons.Hatched boxes indicate untranslated regions. The following designationswere used: splice donor site (S/D), hypoxanthine phosphoribosyltransferase (HPRT), neomycin resistance gene (Neo), vector promoter #1(VP #1), vector promoter #2 (VP#2), and vector promoter #3 (VP#3). Inthe vectors shown in FIG. 21, Neo represents the positive selectablemarker and HPRT represents the negative selectable marker. In re to thesecond vector a third promoter is located upstream of the selectablemarkers. This upstream promoter is operably linked to an exon andunpaired splice donor site. The region designated exon contains atranslation start codon in this example. As described herein, the exonmay encode a methionine residue, a partial signal sequence, a fullsignal secretion sequence, a portion of a protein, or an epitope tag. Inaddition, the codons may be present in any reading frame relative to thesplice donor site. In other vector examples not shown, the regiondesignated exon lacks a translation start codon.

FIG. 22. Examples of transcripts produced by a dual positive/negativeselectable marker vector integrated into a host cell genome upstream ofa multi-exon endogenous gene. Each horizontal line represents a DNAmolecule. Vertical lines running through the DNA strand mark theupstream and downstream vector/cellular genome boundaries. The arrowsdenote promoter sequences located on the DNA molecule, and face in thedirection of transcription. Transcribed regions include all sequenceslocated downstream of each promoter. Boxes indicate exons. Hatched boxesindicate untranslated regions. The endogenous exons are numbered usingroman numerals. The following designations were used: splice donor site(S/D), neomycin resistance gene (Neo), vector promoter #1 (VP#1), vectorpromoter #2 (VP#2), vector promoter #3 (VP#3), polyadenylation signal(pA), and endogenous promoter (EP). Following integration, vectorpromoter #1 expresses a chimeric transcript containing the Neo genelinked to the genomic sequences downstream of the integration site,including the processed (spliced) exons from the endogenous gene. Sincetranscript #1 contains a poly (A) signal from the endogenous gene, theNeo gene product will be efficiently produced, thereby conferring drugresistance on the cell. In addition to transcript #1, the integratedvector will generate a second transcript, designated transcript. #2,originating from vector promoter #2. In this example, the vector hasintegrated upstream of a multi-exon gene. Since multi exon genes containsplice acceptor sites at the 5′ end of each exon, the HPRT gene will beremoved from transcript #2 in cells in which the vector has integratednear, and transcriptionally activated, a multi-exon gene. As a result,cells containing activated multi-exon genes may be isolated by selectingwith G418 and 8-Azaguanine 6-Thioguanine (AgThg). Thus, cells containinga vector integrated near single exon genes will survive double selectionwith G418 and AgThg. These cells can be used to efficiently isolate theactivated multi-exon genes using methods described herein. In additionto transcripts #1 and #2, a third transcript, designated transcript #3is produced from the integrated vector. Transcript #3, originating fromvector promoter #3, contains an exonic sequence suitable for directingprotein expression from the endogenous gene. This occurs followingsplicing from the first splice donor site downstream of promoter #3 tothe first downstream splice acceptor site from the endogenous gene. Inaddition to directing protein expression, transcript #3, and/ortranscripts #1 and/or #2, can be isolated for gene discovery purposesusing the methods described herein.

FIGS. 23A-23D. Example of a multi-Promoter/Activation Exon Vector. Eachvector is illustrated schematically in its linearized form. Eachhorizontal line represents a DNA molecule. The arrows denote promotersequences. Boxes indicate exons. Hatched boxes indicate untranslatedregions. It is understood that the exons on these vectors may beuntranslated, or may contain a start codon and additional codons asdescribed herein The following designations were used: splice donor site(S/D), vector promoter #1 (VP #1), vector promoter #2 (VP#2), vectorpromoter #3 (VP #3), and vector promoter #4 (VP#4). Individual vectoractivation exons are designated A, B, C, and D. Each activation exon maycontain a different structure. The structure of each activation exon andits flanking intron are shown below. It is understood, however, that anyactivation exon described herein, may be used on these vectors, in anycombination and/or order, including exons that encode signal sequences,partial signal sequences, epitope tags, proteins, portions of proteins,and protein motifs. Any of the exons may lack a start codon. Inaddition, while not illustrated in these examples, these vectors maycontain a selectable marker and/or an amplifiable marker. The selectablemarker may contain a poly (A) signal or a splice donor site. Whenpresent, the splice donor site may be located upstream or downstream ofthe selectable marker. Alternatively, the selectable marker may not beoperably linked to a poly (A) signal and/or a splice donor site.

FIG. 24. Examples of transcripts produced from amulti-Promoter/Activation Exon Vector upon integration into a host cellgenome upstream of an endogenous gene. Each horizontal line represents aDNA molecule. Vertical lines running through the DNA strand mark theupstream and downstream vector/cellular genome boundaries. The arrowsdenote promoter sequences located on the DNA molecule, and face in thedirection of transcription. Transcribed regions include all sequenceslocated downstream of each promoter. Boxes indicate exons. Hatched boxesindicate untranslated regions. The endogenous exons are numbered usingroman numerals. The following designations were used: splice donor site(S/D), vector promoter #1 (VP #1), vector promoter #2 (VP#2), vectorpromoter #3 (VP #3), vector promoter #4 (VP#4), endogenous promoter(EP), and polyadenylation signal (pA). Individual vector activationexons are designated A, B, C, and D. Following integration, each vectorencoded promoter is capable of producing a different transcript. Eachtranscript contains a different activation exon joined to the firstdownstream splice acceptor site from an endogenous gene (exon II in thisexample). Individual activation exons are designated by (A), (B), (C),or (D). Endogenous exons are designated by (I), (II), (III), or (IV).Generally, the coding sequence and/or reading frames, if present, aredifferent among the activation exons. While four activation exons areillustrated in this example, any number of activation exons may bepresent on the integrated vector.

FIGS. 25A-25D. Examples of activation vectors useful for detection ofprotein—protein interactions. Each vector is illustrated schematicallyin its linearized form. Each horizontal line represents a DNA molecule.The arrows denote promoter sequences. Boxes indicate exons. Hatchedboxes indicate untranslated regions. The following designations wereused: splice donor site (S/D), neomycin resistance gene (Neo). It isalso recognized that the DNA binding domain and the Activation domainmay be encoded in any reading frame (relative to the splice donor site),allowing activation of endogenous genes with different reading frames.

FIG. 26. Schematic illustration depicting one approach to detectingprotein-protein interactions using the vectors shown in FIG. 25. Eachhorizontal line represents a DNA molecule. Vertical lines runningthrough the DNA strand mark the upstream and downstream vector/cellulargenome boundaries. The arrows denote promoter sequences located on theDNA molecule, and face in the direction of transcription. Transcribedregions include all sequences located downstream of each promoter. Boxesindicate exons. Hatched boxes indicate untranslated regions. Theendogenous exons are numbered using roman numerals. The followingdesignations were used: splice donor site (S/D), binding domain (ED),activation domain (AD), recognition sequence (RS), and polyadenylationsignal (pA). The binding domain vector is shown integrated into thegenome of a host cell, upstream of an endogenous gene, designated geneA. The activation domain vector is shown integrated into the genome ofthe same host cell upstream of an endogenous gene, designated gene B.Both vectors are integrated into the genome of the same host cell.Following integration, each vector is capable of producing a fusionprotein containing the binding domain (or activation domain, as the casemaybe) and the protein encoded by the downstream endogenous gene. If thebinding domain fusion protein interacts with the activation domainfusion protein, a protein complex will be formed. This complex iscapable of increasing expression of a reporter gene present in the cell.

FIG. 27. Examples of activation vectors useful for in vitro and in vivotransposition. Each vector is illustrated schematically in itslinearized form. Each horizontal line represents a DNA molecule. Thearrows denote promoter sequences. Boxes indicate exons. Hatched boxesindicate untranslated regions. The solid boxes indicate the transposonsignals. It is recognized that there is directionality to the transposonsignals, and that the signals are oriented in the configuration suitablefor the type of transposition reaction (integration, inversion, ordeletion). The following designations were used: splice donor site(S/D), neomycin resistance gene (Neo), dihydrofolate reductase (DHFR),puromycin resistance gene (Puro), poly (A) signal (pA), and the EpsteinBarr Virus origin of replication (ori P). It is also recognized thatactivation exon may be encode amino acids in any reading frame (relativeto the splice donor site), allowing activation of endogenous genes withdifferent reading frames.

FIG. 28. Schematic illustration depicting integration of an activationvector into a cloned genomic DNA fragment by in vitro transposition.Each horizontal line represents a DNA molecule. The cloned genomic DNAis in a BAC vector. The single line represents the genomic DNA and therectangle depicts the BAC vector sequences. The arrows denote promotersequences located on the DNA molecule, and face in the direction oftranscription. Transcribed regions include all sequences locateddownstream of each promoter. The vector activation exon is depicted asan open box. Exons from a gene encoded in the cloned genomic fragmentare depicted as hatched boxes. The solid boxes indicate the transposonsignals. It is recognized that there is directionality to the transposonsignals, and that the signals are oriented in the configuration suitablefor the type of transposition reaction (integration, inversion, ordeletion). The following designations were used: splice donor site(S/D), and polyadenylation signal (pA). To integrate the vector into thegenomic fragment, the activation vector is incubated with the clonedgenomic DNA in the presence of transposase. Following integration of theactivation vector into the genomic fragment, the plasmid may betransfected directly into an appropriate eukaryotic host cell to expressthe gene located downstream of the vector integration site.Alternatively, the BAC plasmid may be transformed into E. coli toproduce larger quantities of plasmid for transfection into theappropriate eukaryotic host cell.

FIGS. 29A-29B. Nucleotide sequence of pRIG14.

FIGS. 30A-30C. Nucleotide sequence of pRIG19.

FIGS. 31A-31C. Nucleotide sequence of pRIG20.

FIGS. 32A-32C. Nucleotide sequence of pRIGad1.

FIGS. 33A-33D. Nucleotide sequence of pRIGbd1.

FIGS. 34A-34B. Nucleotide sequence of pUniBAC.

FIGS. 35A-35B. Nucleotide sequence of pRIG22.

FIG. 36. Schematic diagram of pRIG-TP. The vector is shown in itslinearized form. The horizontal line represents a DNA molecule. Thearrows denote promoters. Open boxes indicate exons. Filled boxesrepresent transposon recombination signals (from Tn5—compatible with thein vitro transposition kit available from Epicentre Technologies). Thefollowing designations were used: splice donor site (S/D), puromycinresistance gene (puro), dihydrofolate reductase gene (DHFR), EpsteinBarr nuclear antigen—1 replication protein (EBNA-1), Epstein Barr virusorigin of replication (ori P), poly (A) signal (pA), and activation exon(AE). It is understood that the activation exon can contain any sequencecapable of directing protein synthesis, including a translation startcodon in any reading frame, a partial secretion signal sequence, anentire secretion signal sequence, an epitope tag, a protein, a portionof a protein, or a protein motif. The activation exon may also lack atranslation start codon.

FIGS. 37A-37C. Nucleotide sequence of pRIG-T.

DETAILED DESCRIPTION OF THE INVENTION

There are great advantages to gene activation by non-homologousrecombination over other gene activation procedures. Unlike previousmethods of protein over-expression, the methods described herein do notrequire that the gene of interest be cloned (isolated from the cell).Nor do they require any knowledge of the DNA sequence or structure ofthe gene to be over-expressed (i.e., the sequence of the ORF, introns,exons, or upstream and downstream regulatory elements) or knowledge of agene's expression patterns (i.e., tissue specificity, developmentalregulation, etc.). Furthermore, the methods do not require any knowledgepertaining to the genomic organization of the gene of interest (i.e.,the intron and exon structure).

The methods of the present invention thus involve vector constructs thatdo not contain target nucleotide sequences for homologous recombination.A target sequence allows homologous recombination of vector DNA withcellular DNA at a predetermined site on the cellular DNA, the sitehaving homology for sequences in the vector, the homologousrecombination at the predetermined site resulting in the introduction ofthe transcriptional regulatory sequence into the genome and thesubsequent endogenous gene activation.

The method of the present invention does not involve integration of thevector at predetermined sites. Instead, the present methods involveintegration of the vector constructs of the invention into cellular DNA(e.g., the cellular genome) by nonhomologous or “illegitimate”recombination, also called “non-targeted gene activation.” In relatedembodiments, the present invention also concerns non-targeted geneactivation. Non-targeted gene activation has a number of importantapplications. First, by activating genes that are not normally expressedin a given cell type, it becomes possible to isolate a cDNA copy ofgenes independent of their normal expression pattern. This facilitatesisolation of genes that are normally expressed in rare cells, duringshort developmental periods, and/or at very low levels. Second, bytranslationally activating genes, it is possible to produce proteinexpression libraries without the need for cloning the full-length cDNA.These libraries can be screened for new enzymes and proteins and/or forinteresting phenotypes resulting from over-expression of an endogenousgene. Third, cell-lines over-expressing a specific protein can becreated and used to produce commercial quantities of protein. Thus,activating endogenous genes provides a powerful approach to discoveringand isolating new genes and proteins, and to producing large amounts ofspecific proteins for commercialization.

The vectors described herein do not contain target sequences. A targetsequence is a sequence on the vector that has homology with a sequenceor sequences within the gene to be activated or upstream of the gene tobe activated, the upstream region being up to and including the firstfunctional splice acceptor site on the same coding strand of the gene ofinterest, and by means of which homology the transcriptional regulatorysequence that activates the gene of interest is integrated into thegenome of the cell containing the gene to be activated. In the case ofan enhancer integration vector for activating an endogenous gene, thevector does not contain homology to any sequence in the genome upstreamor downstream of the gene of interest (or within the gene of interest)for a distance extending as far as enhancer function is operative.

The present methods, therefore, are capable of identifying new genesthat have been or can be missed using conventional and currentlyavailable cloning techniques. By using the constructs and methodologydescribed herein, unknown and/or uncharacterized genes can be rapidlyidentified and over-expressed to produce proteins. The proteins have useas, among other things, human therapeutics and diagnostics and astargets for drug discovery.

The methods are also capable of producing over-expression of knownand/or characterized genes for in vitro or in vivo protein production.

A “known” gene is directed to the level of characterization of a gene.The invention allows expression of genes that have been characterized,as well as expression of genes that have not been characterized.Different levels of characterization are possible. These includedetailed characterization, such as cloning, DNA, RNA, and/or proteinsequencing, and relating the regulation and function of the gene to thecloned sequence (e.g., recognition of promoter and enhancer sequences,functions of the open reading frames, introns, and the like).Characterization can be less detailed, such as having mapped a gene andrelated function, or having a partial amino acid or nucleotide sequence,or having purified a protein and ascertained a function.Characterization may be minimal, as when a nucleotide or amino acidsequence is known or a protein has been isolated but the function isunknown. Alternatively, a function may be known but the associatedprotein or nucleotide sequence is not known or is known but has not beencorrelated to the function. Finally, there may be no characterization inthat both the existence of the gene and its function are not known. Theinvention allows expression of any gene at any of these or otherspecific degrees of characterization.

Many different proteins (also referred to herein interchangeably as“gene products” or “expression products”) can be activated orover-expressed by a single activation construct and in a single set oftransfections. Thus, a single cell or different cells in a set oftransfectants (library) can over-express more than one protein followingtransfection with the same or different constructs. Previous activationmethods require a unique construct to be created for each gene to beactivated.

Further, many different integration sites adjacent to a single gene canbe created and tested simultaneously using a single construct. Thisallows rapid determination of the optimal genomic location of theactivation construct for protein expression.

Using previous methods, the 5′ end of the gene of interest had to beextensively characterized with respect to sequence and structure. Foreach activation construct to be produced, an appropriate targetingsequence had to be isolated. Usually, this must be an isogenic sequenceisolated from the same person or laboratory strain of animal as thecells to be activated. In some cases, this DNA may be 50 kb or more fromthe gene of interest. Thus, production of each targeting constructrequired an arduous amount of cloning and sequencing of the endogenousgene. However, since sequence and structure information is not requiredfor the methods of the present invention, unknown genes and genes withuncharacterized upstream regions can be activated.

This is made possible using in situ gene activation using non-homologousrecombination of exogenous DNA sequences with cellular DNA. Methods andcompositions (e.g., vector constructs) required to accomplish such insitu gene activation using non-homologous recombination are provided bythe present invention.

DNA molecules can recombine to redistribute their genetic content byseveral different and distinct mechanisms, including homologousrecombination, site-specific recombination, andnon-homologous/illegitimate recombination. Homologous recombinationinvolves recombination between stretches of DNA that are highly similarin sequence. It has been demonstrated that homologous recombinationinvolves pairing between the homologous sequences along their lengthprior to redistribution of the genetic material. The exact site ofcrossover can be at any point in the homologous segments. The efficiencyof recombination is proportional to the length of homologous targetingsequence (Hope, Development 113:399(1991); Reddy et al, J. Virol.65:1507 (1991)), the degree of sequence identity between the tworecombining sequences (von Melchner et al., Genes Dev. 6:919(1992)), andthe ratio of homologous to non-homologous DNA present on the construct(Letson, Genetics 117:759 (1987)).

Site-specific recombination, on the other hand, involves the exchange ofgenetic material at a predetermined site, designated by specific DNAsequences. In this reaction, a protein recombinase binds to therecombination signal sequences, creates a strand scission, andfacilitates DNA strand exchange. Cre/Lox recombination is an example ofsite specific recombination.

Non-homologous/illegitimate recombination, such as that usedadvantageously by the methods of the present invention, involves thejoining (exchange or redistribution) of genetic material that does notshare significant sequence homology and does not occur at site-specificrecombination sequences. Examples of non-homologous recombinationinclude integration of exogenous DNA into chromosomes at non-homologoussites, chromosomal translocations and deletions, DNA end-joining, doublestrand break repair of chromosome ends, bridge-breakage fusion, andconcatemerization of transfected sequences. In most cases,non-homologous recombination is thought to occur through the joining of“free DNA ends.” Free ends are DNA molecules that contain an end capableof being joined to a second DNA end either directly, or following repairor processing. The DNA end may consist of a 5′ overhang, 3′ overhang, orblunt end.

As used herein, retroviral insertion and other transposition reactionsare loosely considered forms of non-homologous recombination. Thesereactions do not involve the use of homology between the recombiningmolecules. Furthermore, unlike site-specific recombination, these typesof recombination reactions do not occur between discrete sites. Instead,a specific protein/DNA complex is required on only one of therecombination partners (i.e., the retrovirus or transposon), with thesecond DNA partner (i.e., the cellular genome) usually being relativelynon-specific. As a result, these “vectors” do not integrate into thecellular genome in a targeted fashion, and therefore they can be used todeliver the activation construct according to the present invention.

Vector constructs useful for the methods described herein ideally maycontain a transcriptional regulatory sequence that undergoesnon-homologous recombination with genomic sequences in a cell toover-express an endogenous gene in that cell. The vector constructs ofthe invention also lack homologous targeting sequences. That is, they donot contain DNA sequences that target host cell DNA and promotehomologous recombination at the target site. Thus, integration of thevector constructs of the present invention into the cellular genomeoccurs by non-homologous recombination, and can lead to over-expressionof a cellular gene via the introduced transcriptional regulatorysequence contained on the integrated vector construct.

The invention is generally directed to methods for over-expressing anendogenous gene in a cell, comprising introducing a vector containing atranscriptional regulatory sequence into the cell, allowing the vectorto integrate into the genome of the cell by non-homologousrecombination, and allowing over-expression of the endogenous gene inthe cell. The method does not require previous knowledge of the sequenceof the endogenous gene or even of the existence of the gene. Where thesequence of the gene to be activated is known, however, the constructscan be engineered to contain the proper configuration of vector elements(e.g., location of the start codon, addition of codons present in thefirst exon of the endogenous gene, and the proper reading frame) toachieve maximal over expression and/or the appropriate protein sequence.

In certain embodiments of the invention, the cell containing the vectormay be screened for expression of the gene.

The cell over-expressing the gene can be cultured in vitro underconditions favoring the production, by the cell, of desired amounts ofthe gene product of the endogenous gene that has been activated or whoseexpression has been increased. If desired, the gene product can then beisolated or purified to use, for example, in protein therapy or drugdiscovery.

Alternatively, the cell expressing the desired gene product can beallowed to express the gene product in vivo.

The vector construct can consist essentially of the transcriptionalregulatory sequence.

Alternatively, the vector construct can consist essentially of thetranscriptional regulatory sequence and one or more amplifiable markers.

The invention, therefore, is also directed to methods forover-expressing an endogenous gene in a cell, comprising introducing avector containing a transcriptional regulatory sequence and anamplifiable marker into the cell, allowing the vector to integrate intothe genome of the cell by non-homologous recombination, and allowingover-expression of the endogenous gene in the cell.

The cell containing the vector is screened for over-expression of thegene.

The cell over-expressing the gene is cultured such that amplification ofthe endogenous gene is obtained. The cell can then be cultured in vitroso as to produce desired amounts of the gene product of the amplifiedendogenous gene that has been activated or whose expression has beenincreased. The gene product can then be isolated and purified.

Alternatively, following amplification, the cell can be allowed toexpress the endogenous gene and produce desired amounts of the geneproduct in vivo.

The vector construct can consist essentially of the transcriptionalregulatory sequence and the splice donor sequence.

The invention, therefore, is also directed to methods forover-expressing an endogenous gene in a cell comprising introducing avector containing a transcriptional regulatory sequence and an unpairedsplice donor sequence into the cell, allowing the vector to integrateinto the genome of the cell by non-homologous recombination, andallowing over-expression of the endogenous gene in the cell.

The cell containing the vector is screened for expression of the gene.

The cell over-expressing the gene can be cultured in vitro so as toproduce desirable amounts of the gene product of the endogenous genewhose expression has been activated or increased. The gene product canthen be isolated and purified.

Alternatively, the cell can be allowed to express the desired geneproduct in vivo.

The vector construct can consist essentially of a transcriptionalregulatory sequence operably linked to an unpaired splice donor sequenceand also containing an amplifiable marker.

Other activation vectors include constructs with a transcriptionalregulatory sequence and an exonic sequence containing a start codon; atranscriptional regulatory sequence and an exonic sequence containing atranslational start codon and a secretion signal sequence; constructswith a transcriptional regulatory sequence and an exonic sequencecontaining a translation start codon, and an epitope tag; constructscontaining a transcriptional regulatory sequence and an exonic sequencecontaining a translational start codon, a signal sequence and an epitopetag; constructs containing a transcriptional regulatory sequence and anexonic sequence with a translation start codon, a signal secretionsequence, an epitope tag, and a sequence-specific protease site. In eachof the above constructs, the exon on the construct is locatedimmediately upstream of an unpaired splice donor site.

The constructs can also contain a regulatory sequence, a selectablemarker lacking a poly(A) signal, an internal ribosome entry site (ires),and an unpaired splice donor site (FIG. 4). A start codon, signalsecretion sequence, epitope tag, and/or a protease cleavage site mayoptionally be included between the ires and the unpaired splice donorsequence. When this construct integrates upstream of a gene, theselectable marker will be efficiently expressed since a poly(A) sitewill be supplied by the endogenous gene. In addition the downstream genewill also be expressed since the ires will allow protein translation toinitiate at the downstream open reading frame (i.e. the endogenousgene). Thus, the message produced by this activation construct will bepolycistronic. The advantage of this construct is that integrationevents that do not occur near genes and in the proper orientation willnot produce a drug resistant colony. The reason for this is that withouta poly(A) tail (supplied by the endogenous gene), the neomycinresistance gene will not express efficiently. By reducing the number ofnonproductive integration events, the complexity of the library can bereduced without affecting its coverage (the number of genes activated),and this will facilitate the screening process.

In another embodiment of this construct, cre-lox recombination sequencescan be included between the regulatory sequence and the neo start codonand between the ires and the unpaired splice donor site (between theires and the start codon, if present). Following isolation of cells thathave activated the gene of interest, the neo gene and ires can beremoved by transfecting the cells with a plasmid encoding the crerecombinase. This would eliminate the production of the polycistronicmessage and allow the endogenous gene to be expressed directly from theregulatory sequence on the integrated activation construct. Use of Crerecombination to facilitate deletion of genetic elements from mammalianchromosomes has been described (Gu et al., Science 265:103 (1994);Sauer, Meth. Enzymology 225:890-900 (1993)).

Thus, constructs useful in the methods described herein include, but arenot limited to, the following (See also FIGS. 1-4):

1) Construct with a regulatory sequence and an exon lacking atranslation start codon.

2) Construct with a regulatory sequence and an exon lacking atranslation start codon followed by a splice donor site.

3) Construct with a regulatory sequence and an exon containing atranslation start codon in reading frame 1 (relative to the splice donorsite), followed by an unpaired splice donor site.

4) Construct with a regulatory sequence and an exon containing atranslation start codon in reading frame 2 (relative to the splice donorsite), followed by an unpaired splice donor site.

5) Construct with a regulatory sequence and an exon containing atranslation start codon in reading frame 3 (relative to the splice donorsite), followed by an unpaired splice donor site.

6) Construct with a regulatory sequence and an exon containing atranslation start codon and a signal secretion sequence in reading frame1 (relative to the splice donor site), followed by an unpaired splicedonor site.

7) Construct with a regulatory sequence and an exon containing atranslation start codon and a signal secretion sequence in reading frame2 (relative to the splice donor site), followed by an unpaired splicedonor site.

8) Construct with a regulatory sequence and an exon containing atranslation start codon and a signal secretion sequence in reading frame3 (relative to the splice donor site), followed by an unpaired splicedonor site.

9) Construct with a regulatory sequence and an exon containing (from 5′to 3′) a translation start codon and an epitope tag in reading frame 1(relative to the splice donor site), followed by an unpaired splicedonor site.

10) Construct with a regulatory sequence and an exon containing (from 5′to 3′) a translation start codon and an epitope tag in reading frame 2(relative to the splice donor site), followed by an unpaired splicedonor site.

11) Construct with a regulatory sequence and an exon containing (from 5′to 3′) a translation start codon and an epitope tag in reading frame 3(relative to the splice donor site), followed by an unpaired splicedonor site.

12) Construct with a regulatory sequence and an exon containing (from 5′to 3′) a translation start codon, a signal secretion sequence, and anepitope tag in reading frame 1 (relative to the splice donor site),followed by an unpaired splice donor site.

13) Construct with a regulatory sequence and an exon containing (from 5′to 3′) a translation start codon, a signal secretion sequence, and anepitope tag in reading frame 2 (relative to the splice donor site),followed by an unpaired splice donor site.

14) Construct with a regulatory sequence and an exon containing (from 5′to 3′) a translation start codon, a signal secretion sequence, and anepitope tag in reading frame 3 (relative to the splice donor site),followed by an unpaired splice donor site.

15) Construct with a regulatory sequence and an exon containing (from 5′to 3′) a translation start codon, a signal secretion sequence, anepitope tag, and a sequence specific protease site in reading frame 1(relative to the splice donor site), followed by an unpaired splicedonor site.

16) Construct with a regulatory sequence and an exon containing (from 5′to 3′) a translation start codon, a signal secretion sequence, anepitope tag, and a sequence specific protease site in reading frame 2(relative to the splice donor site), followed by an unpaired splicedonor site.

17) Construct with a regulatory sequence and an exon containing (from 5′to 3′) a translation start codon, a signal secretion sequence, anepitope tag, and a sequence specific protease site in reading frame 3(relative to the splice donor site), followed by an unpaired splicedonor site.

18) Construct with a regulatory sequence linked to a selectable marker,followed by an internal ribosome entry site, and an unpaired splicedonor site.

19) Construct 18 in which a cre/lox recombination signal is locatedbetween a) the regulatory sequence and the open reading frame of theselectable marker and b) between the ires and the unpaired splice donorsite.

20) Construct with a regulatory sequence operably linked to an exoncontaining green fluorescent protein lacking a stop codon, followed byan unpaired splice donor site.

It is to be understood, however, that any vector used in the methodsdescribed herein can include one or more (i.e., one, two, three, four,five, or more, and most preferably one or two) amplifiable markers.Accordingly, methods can include a step in which the endogenous gene isamplified. Placement of one or more amplifiable markers on theactivation construct results in the juxtaposition of the gene ofinterest and the one or more amplifiable markers in the activated cell.Once the activated cell has been isolated, expression can be furtherincreased by selecting for cells containing an increased copy number ofthe locus containing both the gene of interest and the activationconstruct. This can be accomplished by selection methods known in theart, for example by culturing cells in selective culture mediacontaining one or more selection agents that are specific for the one ormore amplifiable markers contained on the genetic construct or vector.

Following activation of an endogenous gene by nonhomologous integrationof any of the vectors described above, the expression of the endogenousgene may be further increased by selecting for increased copies of theamplifiable marker(s) located on the integrated vector. While such anapproach may be accomplished using one amplifiable marker on theintegrated vector, in an alternative embodiment the invention providessuch methods wherein two or more (i.e., two, three, four, five, or more,and most preferably two) amplifiable markers may be included on thevector to facilitate more efficient selection of cells that haveamplified the vector and flanking gene of interest. This approach isparticularly useful in cells that have a functional endogenous copy ofone or more of the amplifiable marker(s) that are contained on thevector, since the selection procedure can result in isolation of cellsthat have incorrectly amplified the endogenous amplifiable marker(s)rather than the vector-encoded amplifiable marker(s). This approach isalso useful to select against cells that develop resistance to theselective agent by mechanisms that do not involve gene amplification.The approach using two or more amplifiable markers is advantageous inthese situations because the probability of a cell developing resistanceto two or more selective agents (resistance to which is encoded by twoor more amplifiable markers) without amplifying the integrated vectorand flanking gene of interest is significantly lower than theprobability of the cell developing resistance to any single selectiveagent. Thus, by selecting for two or more vector encoded amplifiablemarkers, either simultaneously or sequentially, a greater percentage ofcells that are ultimately isolated will contain the amplified vector andgene of interest.

Thus, in another embodiment, the vectors of the invention may containtwo or more (i.e., two, three, four, five, or more, and most preferablytwo) amplifiable markers. This approach allows more efficientamplification of the vector sequences and adjacent gene of interestfollowing activation of expression.

Examples of amplifiable markers that may be used constructing thepresent vectors include, but are not limited to, dihydrofolatereductase, adenosine deaminase, aspartate transcarbamylase,dihydro-orotase, and carbamyl phosphate synthase.

It is also understood that any of the constructs described herein maycontain a eukaryotic viral origin of replication, either in place of, orin conjunction with an amplifiable marker. The presence of the viralorigin of replication allows the integrated vector and adjacentendogenous gene to be isolated as an episome and/or amplified to highcopy number upon introduction of the appropriate viral replicationprotein. Examples of useful viral origins include, but are not limitedto, SV40 ori and EBV ori P.

The invention also encompasses embodiments in which the constructsdisclosed herein consist essentially of the components specificallydescribed for these constructs. It is also understood that the aboveconstructs are examples of constructs useful in the methods describedherein, but that the invention encompasses functional equivalents ofsuch constructs.

The term “vector” is understood to generally refer to the vehicle bywhich the nucleotide sequence is introduced into the cell. It is notintended to be limited to any specific sequence. The vector could itselfbe the nucleotide sequence that activates the endogenous gene or couldcontain the sequence that activates the endogenous gene. Thus, thevector could be simply a linear or circular polynucleotide containingessentially only those sequences necessary for activation, or could bethese sequences in a larger polynucleotide or other construct such as aDNA or RNA viral genome, a whole virion, or other biological constructused to introduce the critical nucleotide sequences into a cell. It isalso understood that the phrase “vector construct” or the term“construct” may be used interchangeably with the term “vector” herein.

The vector can contain DNA sequences that exist in nature or that havebeen created by genetic engineering or synthetic processes.

The construct, upon nonhomologous integration into the genome of a cell,can activate expression of an endogenous gene. Expression of theendogenous gene may result in production of full length protein, or inproduction of a truncated biologically active form of the endogenousprotein, depending on the integration site (e.g., upstream region versusintron 2). The activated gene may be a known gene (e.g., previouslycloned or characterized) or unknown gene (previously not cloned orcharacterized). The function of the gene may be known or unknown.

Examples of proteins with known activities include, but are not limitedto, cytokines, growth factors, neurotransmitters, enzymes, structuralproteins, cell surface receptors, intracellular receptors, hormones,antibodies, and transcription factors. Specific examples of knownproteins that can be produced by this method include, but are notlimited to, erythropoietin, insulin, growth hormone, glucocerebrosidase,tissue plasminogen activator, granulocyte-colony stimulating factor(G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF),macrophage colony-stimulating factor (M-CSF) interferon α, interferon β,interferon γ, interleukin-2, interleukin-3, interleukin-4,interleukin-6, interleukin-8, interleukin-10, interleukin-11,interleukin-12, interleukin-13, interleukin-14, TGF-β, blood clottingfactor V, blood clotting factor VII, blood clotting factor VIII, bloodclotting factor IX, blood clotting factor X, TSH-β, bone growthfactor-2, bone growth factor-7, tumor necrosis factor, alpha-1antitrypsin, anti-thrombin III, leukemia inhibitory factor, glucagon,Protein C, protein kinase C, stem cell factor, follicle stimulatinghormone β, urokinase, nerve growth factors, insulin-like growth factors,insulinotropin, parathyroid hormone, lactoferrin, complement inhibitors,platelet derived growth factor, keratinocyte growth factor, hepatocytegrowth factor, endothelial cell growth factor, neurotropin-3,thrombopoietin, chorionic gonadotropin, thrombomodulin, alphaglucosidase, epidermal growth factor, and fibroblast growth factor. Theinvention also allows the activation of a variety of genes expressingtransmembrane proteins, and production and isolation of such proteins,including but not limited to cell surface receptors for growth factors,hormones, neurotransmitters and cytokines such as those described above,transmembrane ion channels, cholesterol receptors, receptors forlipoproteins (including LDLs and HDLs) and other lipid moieties,integrins and other extracellular matrix receptors, cytoskeletalanchoring proteins, immunoglobulin receptors, CD antigens (includingCD2, CD3, CD4, CD8, and CD34 antigens), and other cell surfacetransmembrane structural and functional proteins that are known in theart. As one of ordinary skill will appreciate, other cellular proteinsand receptors that are known in the art may also be produced by themethods of the invention.

One of the advantages of the method described herein is that virtuallyany gene can be activated. However, since genes have different genomicstructures, including different intronlexon boundaries and locations ofstart codons, a variety of activation constructs is provided to activatethe maximum number of different genes within a population of cells.

These constructs can be transfected separately into cells to producelibraries. Each library contains cells with a unique set of activatedgenes. Some genes will be activated by several different activationconstructs. In addition, portions of a gene can be activated to producetruncated, biologically active proteins. Truncated proteins can beproduced, for example, by integration of an activation construct intointrons or exons in the middle of an endogenous gene rather thanupstream of the second exon.

Use of different constructs also allows the activated gene to bemodified to contain new sequences. For example, a secretion signalsequence can be included on the activation construct to facilitate thesecretion of the activated gene. In some cases, depending on theintron/exon structure or the gene of interest, the secretion signalsequence can replace all or part of the signal sequence of theendogenous gene. In other cases, the signal sequence will allow aprotein which is normally located intracellularly to be secreted.

The regulatory sequence on the vector can be a constitutive promoter.Alternatively, the promoter may be inducible. Use of inducible promoterswill allow low basal levels of activated protein to be produced by thecell during routine culturing and expansion. The cells may then beinduced to produce large amounts of the desired proteins, for example,during manufacturing or screening. Examples of inducible promotersinclude, but are not limited to, the tetracycline inducible promoter andthe metallothionein promoter.

In preferred embodiments of the invention, the regulatory sequence onthe vectors of the invention may be a promoter, an enhancer, or arepressor, any of which may be tissue specific.

The regulatory sequence on the vector can be isolated from cellular orviral genomes. Examples of cellular regulatory sequences include, butare not limited to, regulatory elements from the actin gene,metallothionein I gene, immunoglobulin genes, casein I gene, serumalbumin gene, collagen gene, globin genes, laminin gene, spectrin gene,ankyrin gene, sodium/potassium ATPase gene, and tubulin gene. Examplesof viral regulatory sequences include, but are not limited to,regulatory elements from Cytomegalovirus (CMV) immediate early gene,adenovirus late genes, SV40 genes, retroviral LTRs, and Herpesvirusgenes. Typically, regulatory sequences contain binding sites fortranscription factors such as NF-kB, SP-1, TATA binding protein, AP-1,and CAAT binding protein. Functionally, the regulatory sequence isdefined by its ability to promote, enhance, or otherwise altertranscription of an endogenous gene.

In certain preferred embodiments, the regulatory sequence is a viralpromoter. In particularly preferred embodiments, the promoter is the CMVimmediate early gene promoter. In alternative embodiments, theregulatory element is a cellular, non-viral promoter.

In alternative preferred embodiments, the regulatory element may be ormay contain an enhancer. In particularly preferred such embodiments, theenhancer is the cytomegalovirus immediate early gene enhancer. Inalternative embodiments, the enhancer is a cellular, non-viral enhancer.

In alternative preferred embodiments, the regulatory element may be ormay contain a repressor. In particularly preferred such embodiments, therepressor may be a viral repressor or a cellular, non-viral repressor.

The transcriptional regulatory sequence can also comprise one or morescaffold-attachment regions or matrix attachment sites, negativeregulatory elements, and transcription factor binding sites. Regulatorysequences can also include locus control regions.

The invention also encompasses the use of retrovirus transcriptionalregulatory sequences, e.g., long terminal repeats. Where these are used,however, they are not necessarily linked to any retrovirus sequence thatmaterially affects the function of the transcriptional regulatorysequence as a promoter or enhancer of transcription of the endogenousgene to be activated (i.e., the cellular gene with which thetranscriptional regulatory sequence recombines to activate).

The vector constructs of the invention may also comprise a regulatorysequence which is not operably linked to exonic sequences on the vector.For example, when the regulatory element is an enhancer, it canintegrate near an endogenous gene (e.g., upstream, downstream, or in anintron) and stimulate expression of the gene from its endogenouspromoter. By this mechanism of activation, exonic sequences from thevector are absent in the transcript of the activated gene.

Alternatively, the regulatory element may be operably linked to an exon.The exon may be a naturally occurring sequence or may be non-naturallyoccurring (e.g., produced synthetically). To activate endogenous geneslacking a start codon in their first exon (e.g., follicle stimulatinghormone-β), a start codon is preferably omitted from the exon on thevector. To activate endogenous genes containing a start codon in thefirst exon (e.g., erythropoietin and growth hormone), the exon on thevector preferably contains a start codon, usually ATG and preferably anefficient translation initiation site (Kozak, J. Mol Biol. 196: 947(1987)). The exon may contain additional codons following the startcodon. These codons may be derived from a naturally occurring gene ormay be non-naturally occurring (e.g., synthetic). The codons may be thesame as the codons present in the first exon of the endogenous gene tobe activated. Alternatively, the codons may be different than the codonspresent in the first exon of the endogenous gene. For example, thecodons may encode an epitope tag, signal secretion sequence,transmembrane domain, selectable marker, or screenable marker.Optionally, an unpaired splice donor site may be present immediately 3′of the exonic sequence. When the structure of the gene to be activatedis known, the splice donor site should be placed adjacent to the vectorexon in a location such that the codons in the vector will be in framewith the codons of the second exon of the endogenous gene followingsplicing. When the structure of the endogenous gene to be activated isnot known, separate constructs, each containing a different readingframe, are used.

Operably linked is defined as a configuration that allows transcriptionthrough the designated sequence(s). For example, a regulatory sequencethat is operably linked to an exonic sequence indicates that the exonicsequence is transcribed. When a start codon is present on the vector,operably linked also indicates that the open reading frame from thevector exon is in frame with the open reading frame of the endogenousgene. Following nonhomologous integration, the regulatory sequence(e.g., a promoter) on the vector becomes operably linked to anendogenous gene and facilitates transcription initiation, at a sitegenerally referred to as a CAP site. Transcription proceeds through theexonic elements on the vector (and, if present, through the start codon,open reading frame, and/or unpaired splice donor site), and through theendogenous gene. The primary transcript produced by this operablelinkage is spliced to create a chimeric transcript containing exonicsequences from both the vector and the endogenous gene. This transcriptis capable of producing the endogenous protein when translated.

An exon or “exonic sequence” is defined as any transcribed sequence thatis present in the mature RNA molecule. The exon on the vector maycontain untranslated sequences, for example, a 5′ untranslated region.Alternatively, or in conjunction with the untranslated sequences, theexon may contain coding sequences such as a start codon and open readingframe. The open reading frame can encode naturally occurring amino acidsequences or non-naturally occurring amino acid sequences (e.g.,synthetic codons). The open reading frame may also encode a signalsecretion sequence, epitope tag, exon, selectable marker, screenablemarker, or nucleotides that function to allow the open reading frame tobe preserved when spliced to an endogenous gene.

Splicing of primary transcripts, the process by which introns areremoved, is directed by a splice donor site and a splice acceptor site,located at the 5′ and 3′ ends of introns, respectively. The consensussequence for splice donor sites is (A/C)AG GURAGU (where R represents apurine nucleotide) with nucleotides in positions 1-3 located in the exonand nucleotides GURAGU located in the intron.

An unpaired splice donor site is defined herein as a splice donor sitepresent on the activation construct without a downstream splice acceptorsite. When the vector is integrated by nonhomologous recombination intoa host cell's genome, the unpaired splice donor site becomes paired witha splice acceptor site from an endogenous gene. The splice donor sitefrom the vector, in conjunction with the splice acceptor site from theendogenous gene, will then direct the excision of all of the sequencesbetween the vector splice donor site and the endogenous splice acceptorsite. Excision of these intervening sequences removes sequences thatinterfere with translation of the endogenous protein.

The terms upstream and downstream, as used herein, are intended to meanin the 5′ or in the 3′ direction, respectively, relative to the codingstrand. The term “upstream region” of a gene is defined as the nucleicacid sequence 5′ of its second exon (relative to the coding strand) upto and including the last exon of the first adjacent gene having thesame coding strand. Functionally, the upstream region is any site 5′ ofthe second exon of an endogenous gene capable of allowing anonhomologously integrated vector to become operably linked to theendogenous gene.

The vector construct can contain a selectable marker to facilitate theidentification and isolation of cells containing a nonhomologouslyintegrated activation construct. Examples of selectable markers includegenes encoding neomycin resistance (neo), hypoxanthine phosphoribosyltransferase (HPRT), puromycin (pac), dihydro-orotase glutaminesynthetase (GS), histidine D (his D), carbamyl phosphate synthase (CAD),dihyrofolate reductase (DHFR), multidrug resistance 1 (mdr1), aspartatetranscarbamylase, xanthine-guanine phosphoribosyl transferase (gpt), andadenosine deaminase (ada).

Alternatively, the vector can contain a screenable marker, in place ofor in addition to, the selectable marker. A screenable marker allows thecells containing the vector to be isolated without placing them underdrug or other selective pressures. Examples of screenable markersinclude genes encoding cell surface proteins, fluorescent proteins, andenzymes. The vector containing cells may be isolated, for example, byFACS using fluorescently-tagged antibodies to the cell surface proteinor substrates that can be converted to fluorescent products by a vectorencoded enzyme.

Alternatively, selection can be effected by phenotypic selection for atrait provided by the endogenous gene product. The activation construct,therefore, can lack a selectable marker other than the “marker” providedby the endogenous gene itself. In this embodiment, activated cells canbe selected based on a phenotype conferred by the activated gene.Examples of selectable phenotypes include cellular proliferation, growthfactor independent growth, colony formation, cellular differentiation(e.g., differentiation into a neuronal cell, muscle cell, epithelialcell, etc.), anchorage independent growth, activation of cellularfactors (e.g., kinases, transcription factors, nucleases, etc.),expression of cell surface receptors/proteins, gain or loss of cell—celladhesion, migration, and cellular activation (e.g., resting versusactivated T cells).

A selectable marker may also be omitted from the construct whentransfected cells are screened for gene activation products withoutselecting for the stable integrants. This is particularly useful whenthe efficiency of stable integration is high.

The vector may contain one or more (i.e., one, two, three, four, five,or more, and most preferably one or two) ampliflable markers to allowfor selection of cells containing increased copies of the integratedvector and the adjacent activated endogenous gene. Examples ofamplifiable markers include but are not limited to dihydrofolatereductase (DHFR), adenosine deaminase (ada), dihydro-orotase glutaminesynthetase (GS), and carbamyl phosphate synthase (CAD).

The vector may contain eukaryotic viral origins of replication usefulfor gene amplification. These origins may be present in place of, or inconjunction with, an amplifiable marker.

The vector may also contain genetic elements useful for the propagationof the construct in micro-organisms. Examples of useful genetic elementsinclude microbial origins of replication and antibiotic resistancemarkers.

These vectors, and any of the vectors disclosed herein, and obviousvariants recognized by one of ordinary skill in the art, can be used inany of the methods described herein to form any of the compositionsproducible by those methods.

Nonhomologous integration of the construct into the genome of a cellresults in the operable linkage between the regulatory elements from thevector and the exons from an endogenous gene. In preferred embodiments,the insertion of the vector regulatory sequences is used to upregulateexpression of the endogenous gene. Upregulation of gene expressionincludes converting a transcriptionally silent gene to atranscriptionally active gene. It also includes enhancement of geneexpression for genes that are already transcriptionally active, butproduce protein at levels lower than desired. In other embodiments,expression of the endogenous gene may be affected in other ways such asdownregulation of expression, creation of an inducible phenotype, orchanging the tissue specificity of expression.

According to the invention, in vitro methods of production of a geneexpression product may comprise, for example, (a) introducing a vectorof the invention into a cell; (b) allowing the vector to integrate intothe genome of the cell by non-homologous recombination; (c) allowingover-expression of an endogenous gene in the cell by upregulation of thegene by the transcriptional regulatory sequence contained on the vector;(d) screening the cell for over-expression of the endogenous gene; and(e) culturing the cell under conditions favoring the production of theexpression product of the endogenous gene by the cell. Such in vitromethods of the invention may further comprise isolating the expressionproduct to produce an isolated gene expression product. In such methods,any art-known method of protein isolation may be advantageously used,including but not limited to chromatography (e.g., HPLC, FPLC, LC, ionexchange, affinity, size exclusion, and the like), precipitation (e.g.,ammonium sulfate precipitation, immunoprecipitation, and the like),electrophoresis, and other methods of protein isolation and purificationthat will be familiar to one of ordinary skill in the art.

Analogously, in vivo methods of production of a gene expression productmay comprise, for example, (a) introducing a vector of the inventioninto a cell; (b) allowing the vector to integrate into the genome of thecell by non-homologous recombination; (c) allowing over-expression of anendogenous gene in the cell by upregulation of the gene by thetranscriptional regulatory sequence contained on the vector; (d)screening the cell for over-expression of the endogenous gene; and (e)introducing the isolated and cloned cell into a eukaryote underconditions favoring the over expression of the endogenous gene by thecell in vivo in the eukaryote. According to this aspect of theinvention, any eukaryote may be advantageously used, including fungi(particularly yeasts), plants, and animals, more preferably animals,still more preferably vertebrates, and most preferably mammals,particularly humans. In certain related embodiments, the inventionprovides such methods which further comprise isolating and cloning thecell prior to introducing it into the eukaryote.

As used herein the phrases “conditions favoring the production” of anexpression product, “conditions favoring the over expression” of a gene,and “conditions favoring the activation” of a gene, in a cell or by acell in vitro refer to any and all suitable environmental, physical,nutritional or biochemical parameters that allow, facilitate, or promoteproduction of an expression product, or over expression or activation ofa gene, by a cell in vitro. Such conditions may, of course, include theuse of culture media, incubation, lighting, humidity, etc., that areoptimal or that allow, facilitate, or promote production of anexpression product, or over expression or activation of a gene, by acell in vitro. Analogously, as used herein the phrases “conditionsfavoring the production” of an expression product, “conditions favoringthe over expression” of a gene, and “conditions favoring the activation”of a gene, in a cell or by a cell in vivo refer to any and all suitableenvironmental, physical, nutritional, biochemical, behavioral, genetic,and emotional parameters under which an animal containing a cell ismaintained, that allow, facilitate, or promote production of anexpression product, or over expression or activation of a gene, by acell in a eukaryote in vivo. Whether a given set of conditions arefavorable for gene expression, activation, or over expression, in vitroor in vivo, may be determined by one of ordinary skill using thescreening methods described and exemplified below, or other methods formeasuring gene expression, activation, or over expression that areroutine in the art.

As used herein, the phrase “activating an endogenous gene” meansinducing the production of a transcript encoding the endogenous gene atlevels higher than those normally found in the cell containing theendogenous gene. In some applications, “activating an endogenous gene”may also mean producing the protein, or a portion of the protein,encoded by the endogenous gene at levels higher than those normallyfound in the cell containing the endogenous gene.

The invention also encompasses cells made by any of the above methods.The invention encompasses cells containing the vector constructs, cellsin which the vector constructs have integrated, and cells which areover-expressing desired gene products from an endogenous gene,over-expression being driven by the introduced transcriptionalregulatory sequence.

Cells used in this invention can be derived from any eukaryotic speciesand can be primary, secondary, or immortalized. Furthermore, the cellscan be derived from any tissue in the organism. Examples of usefultissues from which cells can be isolated and activated include, but arenot limited to, liver, kidney, spleen, bone marrow, thymus, heart,muscle, lung, brain, testes, ovary, islet, intestinal, bone marrow,skin, bone, gall bladder, prostate, bladder, embryos, and the immune andhematopoietic systems. Cell types include fibroblast, epithelial,neuronal, stem, and follicular. However, any cell or cell type can beused to activate gene expression using this invention.

The methods can be carried out in any cell of eukaryotic origin, such asfungal, plant or animal. Preferred embodiments include vertebrates andparticularly mammals, and more particularly, humans.

The construct can be integrated into primary, secondary, or immortalizedcells. Primary cells are cells that have been isolated from a vertebrateand have not been passaged. Secondary cells are primary cells that havebeen passaged, but are not immortalized. Immortalized cells are celllines that can be passaged, apparently indefinitely.

In preferred embodiments, the cells are immortalized cell lines.Examples of immortalized cell lines include, but are not limited to,HT1080, HeLa, Jurkat, 293 cells, KB carcinoma, T84 colonic epithelialcell line, Raji, Hep G2 or Hep 3B hepatoma cell lines, A2058 melanoma,U937 lymphoma, and WI38 fibroblast cell line, somatic cell hybrids, andhybridomas.

Cells used in this invention can be derived from any eukaryotic species,including but not limited to mammalian cells (such as rat, mouse,bovine, porcine, sheep, goat, and human), avian cells, fish cells,amphibian cells, reptilian cells, plant cells, and yeast cells.Preferably, over expression of an endogenous gene or gene product from aparticular species is accomplished by activating gene expression in acell from that species. For example, to overexpress endogenous humanproteins, human cells are used. Similarly, to overexpress endogenousbovine proteins, for example bovine growth hormone, bovine cells areused.

The cells can be derived from any tissue in the eukaryotic organism.Examples of useful vertebrate tissues from which cells can be isolatedand activated include, but are not limited to, liver, kidney, spleen,bone marrow, thymus, heart, muscle, lung, brain, immune system(including lymphatic), testes, ovary, islet, intestinal, stomach, bonemarrow, skin, bone, gall bladder, prostate, bladder, zygotes, embryos,and hematopoietic tissue. Useful vertebrate cell types include, but arenot limited to, fibroblasts, epithelial cells, neuronal cells, germcells (i.e., spermatocytes/spermatozoa and oocytes), stem cells, andfollicular cells. Examples of plant tissues from which cells can beisolated and activated include, but are not limited to, leaf tissue,ovary tissue, stamen tissue, pistil tissue, root tissue, tubers,gametes, seeds, embryos, and the like. One of ordinary skill willappreciate, however, that any eukaryotic cell or cell type can be usedto activate gene expression using the present invention.

Any of the cells produced by any of the methods described are useful forscreening for expression of a desired gene product and for providingdesired amounts of a gene product that is over-expressed in the cell.The cells can be isolated and cloned.

Cells produced by this method can be used to produce protein in vitro(e.g., for use as a protein therapeutic) or in vivo (e.g., for use incell therapy).

Commercial growth and production conditions often vary from theconditions used to grow and prepare cells for analytical use (e.g.,cloning, protein or nucleic acid sequencing, raising antibodies, X-raycrystallography analysis, enzymatic analysis, and the like). Scale up ofcells for growth in roller bottles involves increase in the surface areaon which cells can attach. Microcarrier beads are, therefore, oftenadded to increase the surface area for commercial growth Scale up ofcells in spinner culture may involve large increases in volume. Fiveliters or greater can be required for both microcarrier and spinnergrowth. Depending on the inherent potency (specific activity) of theprotein of interest, the volume can be as low as 1-10 liters. 10-15liters is more common. However, up to 50-100 liters may be necessary andvolume can be as high as 10,000-15,000 liters. In some cases, highervolumes may be required. Cells can also be grown in large numbers of Tflasks, for example 50-100.

Despite growth conditions, protein purification on a commercial scalecan also vary considerably from purification for analytic purposes.Protein purification in a commercial practical context can be initiallythe mass equivalent of 10 liters of cells at approximately 10⁴ cells/ml.Cell mass equivalent to begin protein purification can also be as highas 10 liters of cells at up to 10⁶ or 10⁷ cells/ml. As one of ordinaryskill will appreciate, however, a higher or lower initial cell massequivalent may also be advantageously used in the present methods.

Another commercial growth condition, especially when the ultimateproduct is used clinically, is cell growth in serum-free medium, bywhich is intended medium containing no serum or not in amounts that arerequired for cell growth. This obviously avoids the undesiredco-purification of toxic contaminants (e.g., viruses) or other types ofcontaminants, for example, proteins that would complicate purification.Serum-free media for growth of cells, commercial sources for such media,and methods for cultivation of cells in serum-free media, are well-knownto those of ordinary skill in the art.

A single cell made by the methods described above can over-express asingle gene or more than one gene. More than one gene can be activatedby the integration of a single construct or by the integration ofmultiple constructs in the same cell (i.e., more than one type ofconstruct). Therefore, a cell can contain only one type of vectorconstruct or different types of constructs, each capable of activatingan endogenous gene.

The invention is also directed to methods for making the cells describedabove by one or more of the following: introducing one or more of thevector constructs; allowing the introduced construct(s) to integrateinto the genome of the cell by non-homologous recombination; allowingover-expression of one or more endogenous genes in the cell; andisolating and cloning the cell.

The term “transfection” has been used herein for convenience whendiscussing introducing a polynucleotide into a cell. However, it is tobe understood that the specific use of this term has been applied togenerally refer to the introduction of the polynucleotide into a celland is also intended to refer to the introduction by other methodsdescribed herein such as electroporation, liposome-mediatedintroduction, retrovirus-mediated introduction, and the like (as well asaccording to its own specific meaning).

The vector can be introduced into the cell by a number of methods knownin the art. These include, but are not limited to, electroporation,calcium phosphate precipitation, DEAE dextran, lipofection, and receptormediated endocytosis, polybrene, particle bombardment, andmicroinjection. Alternatively, the vector can be delivered to the cellas a viral particle (either replication competent or deficient).Examples of viruses useful for the delivery of nucleic acid include, butare not limited to, adenoviruses, adeno-associated viruses,retroviruses, Herpesviruseses, and vaccinia viruses. Other virusessuitable for delivery of nucleic acid molecules into cells that areknown to one of ordinary skill may be equivalently used in the presentmethods.

Following transfection, the cells are cultured under conditions, asknown in the art, suitable for nonhomologous integration between thevector and the host cell's genome. Cells containing the nonhomologouslyintegrated vector can be further cultured under conditions, as known inthe art, allowing expression of activated endogenous genes.

The vector construct can be introduced into cells on a single DNAconstruct or on separate constructs and allowed to concatemerize.

Whereas in preferred embodiments, the vector construct is adouble-stranded DNA vector construct, vector constructs also includesingle-stranded DNA, combinations of single- and double-stranded DNA,single-stranded RNA, double-stranded RNA, and combinations of single-and double-stranded RNA. Thus, for example, the vector construct couldbe single-stranded RNA which is converted to cDNA by reversetranscriptase, the cDNA converted to double-stranded DNA, and thedouble-stranded DNA ultimately recombining with the host cell genome.

In preferred embodiments, the constructs are linearized prior tointroduction into the cell. Linearization of the activation constructcreates free DNA ends capable of reacting with chromosomal ends duringthe integration process. In general, the construct is linearizeddownstream of the regulatory element (and exonic and splice donorsequences, if present). Linearization can be facilitated by, forexample, placing a unique restriction site downstream of the regulatorysequences and treating the construct with the corresponding restrictionenzyme prior to transfection While not required, it is advantageous toplace a “spacer” sequence between the linearization site and theproximal most functional element (e.g., the unpaired splice donor site)on the construct. When present, the spacer sequence protects theimportant functional elements on the vector from exonucleolyticdegradation during the transfection process. The spacer can be composedof any nucleotide sequence that does not change the essential functionsof the vector as described herein.

Circular constructs can also be used to activate endogenous geneexpression. It is known in the art that circular plasmids, upontransfection into cells, can integrate into the host cell genomePresumably, DNA breaks occur in the circular plasmid during thetransfection process, thereby generating free DNA ends capable ofjoining to chromosome ends. Some of these breaks in the construct willoccur in a location that does not destroy essential vector functions(e.g., the break will occur downstream of the regulatory sequence), andtherefore, will allow the construct to be integrated into a chromosomein a configuration capable of activating an endogenous gene. Asdescribed above, spacer sequences may be placed on the construct (e.g.,downstream of the regulatory sequences). During transfection, breaksthat occur in the spacer region will create free ends at a site in theconstruct suitable for activation of an endogenous gene followingintegration into the host cell genome.

The invention also encompasses libraries of cells made by the abovedescribed methods. A library can encompass all of the clones from asingle transfection experiment or a subset of clones from a singletransfection experiment. The subset can over-express the same gene ormore than one gene, for example, a class of genes. The transfection canhave been done with a single type of construct or with more than onetype of construct.

A library can also be formed by combining all of the recombinant cellsfrom two or more transfection experiments, by combining one or moresubsets of cells from a single transfection experiment or by combiningsubsets of cells from separate transfection experiments. The resultinglibrary can express the same gene, or more than one gene, for example, aclass of genes. Again, in each of these individual transfections, aunique construct or more than one construct can be used.

Libraries can be formed from the same cell type or different cell types.

The library can be composed of a single type of cell containing a singletype of activation construct which has been integrated into chromosomesat spontaneous DNA breaks or at breaks generated by radiation,restriction enzymes, and/or DNA breaking agents, applied either together(to the same cells) or separately (applied to individual groups of cellsand then combining the cells together to produce the library). Thelibrary can be composed of multiple types of cells containing a singleor multiple constructs which were integrated into the genome of a celltreated with radiation, restriction enzymes, and/or DNA breaking agents,applied either together (to the same cells) or separately (applied toindividual groups of cells and then combining the cells together toproduce the library).

The invention is also directed to methods for making libraries byselecting various subsets of cells from the same or differenttransfection experiments. For example, all of the cells expressingnuclear factors (as determined by the presence of nuclear greenfluorescent protein in cells transfected with construct 20) can bepooled to create a library of cells with activated nuclear factors.Similarly, cells expressing membrane or secreted proteins can be pooled.Cells can also be grouped by phenotype, for example, growth factorindependent growth, growth factor independent proliferation, colonyformation, cellular differentiation (e.g., differentiation into aneuronal cell, muscle cell, epithelial cell, etc.), anchorageindependent growth, activation of cellular factors (e.g., kinases,transcription factors, nucleases, etc.), gain or loss of cell—celladhesion, migration, or cellular activation (e.g., resting versusactivated T cells).

The invention is also directed to methods of using libraries of cells toover-express an endogenous gene. The library is screened for theexpression of the gene and cells are selected that express the desiredgene product. The cell can then be used to purify the gene product forsubsequent use. Expression of the cell can occur by culturing the cellin vitro or by allowing the cell to express the gene in vivo.

The invention is also directed to methods of using libraries to identifynovel gene and gene products.

The invention is also directed to methods for increasing the efficiencyof gene activation by treating the cells with agents that stimulate oreffect the patterns of non-homologous integration. It has beendemonstrated that gene expression patterns, chromatin structure, andmethylation patterns can differ dramatically from cell type to celltype. Even different cell lines from the same cell type can havesignificant differences. These differences can impact the patterns ofnon-homologous integration by affecting both the DNA breakage patternand the repair process. For example, chromatinized stretches of DNA(characteristics likely associated with inactive genes) may be moreresistant to breakage by restriction enzymes and chemical agents,whereas they may be susceptible to breakage by radiation.

Furthermore, inactive genes can be methylated. In this case, restrictionenzymes that are blocked by CpG methylation will be unable to cleavemethylated sites near the inactive gene, making it more difficult toactivate that gene using methylation-sensitive enzymes. These problemscan be circumvented by creating activation libraries in several celllines using a variety of DNA breakage agents. By doing this, a morecomplete integration pattern can be created and the probability ofactivating a given gene maximized.

The methods of the invention can include introducing double strandbreaks into the DNA of the cell containing the endogenous gene to beover-expressed. These methods introduce double-strand breaks into thegenomic DNA in the cell prior to or simultaneously with vectorintegration. The mechanism of DNA breakage can have a significant effecton the pattern of DNA breaks in the genome. As a result, DNA breaksproduced spontaneously or artificially with radiation, restrictionenzymes, bleomycin, or other breaking agents, can occur in differentlocations.

In order to increase integration efficiency and to improve the randomdistribution of integration sites, cells can be treated with low,intermediate, or high doses of radiation prior to or followingtransfection. By artificially inducing double strand breaks, thetransfected DNA can now integrate into the host cell chromosome as partof the DNA repair process. Normally, creation of double strand breaks toserve as the site of integration is the rate limiting step. Thus, byincreasing chromosome breaks using radiation (or other DNA damagingagents), a larger number of integrants can be obtained in a giventransfection. Furthermore, the mechanism of DNA breakage by radiation isdifferent than by spontaneous breakage.

Radiation can induce DNA breaks directly when a high energy photon hitsthe DNA molecule. Alternatively, radiation can activate compounds in thecell which in turn, react with and break the DNA strand. Spontaneousbreaks, on the other hand, are thought to occur by the interactionbetween reactive compounds produced in the cell (such as superoxides andperoxides) and the DNA molecule. However, DNA in the cell is not presentas a naked, deproteinized polymer, but instead is bound to chromatin andpresent in a condensed state. As a result, some regions are notaccessible to agents in the cell that cause double strand breaks. Thephotons produced by radiation have wave lengths short enough to hithighly condensed regions of DNA, thereby inducing breaks in DNA regionsthat are under represented in spontaneous breaks. Thus, radiation iscapable of creating different DNA breakage patterns, which in turn,should lead to different integration patterns.

As a result, libraries produced using the same activation construct incells with and without radiation treatment will potentially containdifferent sets of activated genes. Finally, radiation treatmentincreases efficiency of nonhomologous integration by up to 5-10 fold,allowing complete libraries to be created using fewer cells. Thus,radiation treatment increases the efficiency of gene activation andgenerates new integration and activation patterns in transfected cells.Useful types of radiation include α, β, γ, x-ray, and ultravioletradiation. Useful doses of radiation vary for different cell types, butin general, dose ranges resulting in cell viabilities of 0.1% to >99%are useful. For HT1080 cells, this corresponds to radiation doses from a¹³⁷Cs source of approximately 0.1 rads to 1000 rads. Other doses mayalso be useful as long as the dose either increases the integrationfrequency or changes the pattern of integration sites.

In addition to radiation, restriction enzymes can be used toartificially induce chromosome breaks in transfected cells. As withradiation, DNA restriction enzymes can create chromosome breaks which,in turn, serve as integration sites for the transfected DNA. This largernumber of DNA breaks increases the overall efficiency of integration ofthe activation construct. Furthermore, the mechanism of breakage byrestriction enzymes differs from that by radiation, the pattern ofchromosome breaks is also likely to be different.

Restriction enzymes are relatively large molecules compared to photonsand small metabolites capable of damaging DNA. As a result, restrictionenzymes will tend to break regions that are less condensed then thegenome as a whole. If the gene of interest lies within an accessibleregion of the genome, then treatment of the cells with a restrictionenzyme can increase the probability of integrating the activationconstruct upstream of the gene of interest. Since restriction enzymesrecognize specific sequences, and since a given restriction site may notlie upstream of the gene of interest, a variety of restriction enzymescan be used. It may also be important to use a variety of restrictionenzymes since each enzyme has different properties (e.g., size,stability, ability to cleave methylated sites, and optimal reactionconditions) that affect which sites in the host chromosome will becleaved. Each enzyme, due to the different distribution of cleavablerestriction sites, will create a different integration pattern.

Therefore, introduction of restriction enzymes (or plasmids capable ofexpressing restriction enzymes) before, during, or after introduction ofthe activation construct will result in the activation of different setsof genes. Finally, restriction enzyme-induced breaks increase theintegration efficiency by up to 5-10 fold (Yorifuji et al., Mut. Res.243:121 (1990)), allowing fewer cells to be transfected to produce acomplete library. Thus, restriction enzymes can be used to create newintegration patterns, allowing activation of genes which failed to beactivated in libraries produced by non-homologous recombination atspontaneous breaks or at other artificially induced breaks.

Restriction enzymes can also be used to bias integration of theactivation construct to a desired site in the genome. For example,several rare restriction enzymes have been described which cleaveeukaryotic DNA every 50-1000 kilobases, on average. If a rarerestriction recognition sequence happens to be located upstream of agene of interest, by introducing the restriction enzyme at the time oftransfection along with the activation construct, DNA breaks can bepreferentially upstream of the gene of interest. These breaks can thenserve as sites for integration of the activation construct. Any enzymecan be that cleaves in an appropriate location in or near the gene ofinterest and its site is under-represented in the rest of the genome orits site is over-represented near genes (e.g., restriction sitescontaining CpG). For genes that have not been previously identified,restriction enzymes with 8 bp recognition sites (e.g., NotI, SfiI, PmeI,SwaI, SseI, SrfI, SgrA1, PacI, AscI, SgfI, and Sse8387I), enzymesrecognizing CpG containing sites (e.g., EagI, Bsi-WI, MluI, and BssHII)and other rare cutting enzymes can be used.

In this way, “biased” libraries can be created which are enriched forcertain types of activated genes. In this respect, restriction enzymesites containing CpG dinucleotides are particularly useful since thesesites are under-represented in the genome at large, but over-representedin the form of CpG islands at the 5′ end of many genes, the verylocation that is useful for gene activation. Enzymes recognizing thesesites, therefore, will preferentially cleave at the 5′ end of genicsequences.

Restriction enzymes can be introduced into the host cell by severalmethods. First, restriction enzymes can be introduced into the cell byelectroporation (Yorifuji et al., Mut. Res. 243:121 (1990); Winegar etal., Mut. Res. 225:49 (1989)). In general, the amount of restrictionenzyme introduced into the cell is proportional to its concentration inthe electroporation media. The pulse conditions must be optimized foreach cell line by adjusting the voltage, capacitance, and resistance.Second, the restriction enzyme can be expressed transiently from aplasmid encoding the enzyme under the control of eukaryotic regulatoryelements. The level of enzyme produced can be controlled by usinginducible promoters, and varying the strength of induction. In somecases, it may be desirable to limit the amount of restriction enzymeproduced (due to its toxicity). In these cases, weak or mutantpromoters, splice sites, translation start codons, and poly(A) tails canbe utilized to lower the amount of restriction enzyme produced. Third,restriction enzymes can be introduced by agents that fuse with orpermeabilize the cell membrane. Liposomes and streptolysin O (Pimplikaret al., J. Cell Biol. 125:1025 (1994)) are examples of this type ofagent. Finally, mechanical perforation (Beckers et al., Cell 50:523-534(1987)) and microinjection can also be used to introduce nucleases andother proteins into cells. However, any method capable of deliveringactive enzymes to a living cell is suitable.

DNA breaks induced by bleomycin and other DNA damaging agents can alsoproduce DNA breakage patterns that are different. Thus, any agent orincubation condition capable of generating double strand breaks in cellsis useful for increasing the efficiency and/or altering the sites ofnon-homologous recombination. Examples of classes of chemical DNAbreaking agents include, but are not limited to, peroxides and otherfree radical generating compounds, alkylating agents, topoisomeraseinhibitors, anti-neoplastic drugs, acids, substituted nucleotides, andenediyne antibiotics.

Specific chemical DNA breaking agents include, but are not limited to,bleomycin, hydrogen peroxide, cumene hydroperoxide, tert-butylhydroperoxide, hypochlorous acid (reacted with aniline, 1-naphthylamineor 1-naphthol), nitric acid, phosphoric acid, doxorubicin,9-deoxydoxorubicin, demethyl-6-deoxyrubicin, 5-iminodaunorubicin,adriamycin, 4′-(9-acridinylamino)methanesulfon-m-anisidide,neocarzinostatin, 8-methoxycaffeine, etoposide, ellipticine,iododeoxyuridine, and bromodeoxyuridine.

It has been shown that DNA repair machinery in the cell can be inducedby pre-exposing the cell to low doses of a DNA breaking agent such asradiation or bleomycin. By pretreating cells with these agentsapproximately 24 hours prior to transfection, the cell will be moreefficient at repairing DNA breaks and integrating DNA followingtransfection. In addition, higher doses of radiation or other DNAbreaking agents can be used since the LD50 (the dose that results inlethality in 50% of the exposed cells) is higher following pretreatment.This allows random activation libraries to be created at multiple dosesand results in a different distribution of integration sites within thehost cell's chromosomes.

Screening

Once an activation library (or libraries) is created, it can be screenedusing a number of assays. Depending on the characteristics of theprotein(s) of interest (e.g., secreted versus intracellular proteins)and the nature of the activation construct used to create the library,any or all of the assays described below can be utilized. Other assayformats can also be used.

ELISA. Activated proteins can be detected using the enzyme4inkedimmunosorbent assay (ELISA). If the activated gene product is secreted,culture supernatants from pools of activation library cells areincubated in wells containing bound antibody specific for the protein ofinterest. If a cell or group of cells has activated the gene ofinterest, then the protein will be secreted into the culture media. Byscreening pools of library clones (the pools can be from 1 to greaterthan 100,000 library members), pools containing a cell(s) that hasactivated the gene of interest can be identified. The cell of interestcan then be purified away from the other library members by sibselection, limiting dilution, or other techniques known in the art. Inaddition to secreted proteins, ELISA can be used to screen for cellsexpressing intracellular and membrane-bound proteins. In these cases,instead of screening culture supernatants, a small number of cells isremoved from the library pool (each cell is represented at least100-1000 times in each pool), lysed, clarified, and added to theantibody-coated wells.

ELISA Spot Assay. ELISA spot are coated with antibodies specific for theprotein of interest. Following coating, the wells are blocked with 1%BSA/PBS for 1 hour at 37° C. Following blocking, 100,000 to 500,000cells from the random activation library are applied to each well(representing ˜10% of the total pool). In general, one pool is appliedto each well. If the frequency of a cell expressing the protein ofinterest is 1 in 10,000 (i.e., the pool consists of 10,000 individualclones, one of which expresses the protein of interest), then plating500,000 cells per well will yield 50 specific cells. Cells are incubatedin the wells at 37° C. for 24 to 48 hours without being moved ordisturbed. At the end of the incubation, the cells are removed and theplate is washed 3 times with PBS/0.05% Tween 20 and 3 times with PBS/1%BSA. Secondary antibodies are applied to the wells at the appropriateconcentration and incubated for 2 hours at room temperature or 16 hoursat 4° C. These antibodies can be biotinylated or labeled directly withhorseradish peroxidase (HRP). The secondary antibodies are removed andthe plate is washed with PBS/1% BSA. The tertiary antibody orstreptavidin labeled with HRP is added and incubated for 1 hour at roomtemperature.

FACS assay. The fluorescence-activated cell sorter (FACS) can be used toscreen the random activation library in a number of ways. If the gene ofinterest encodes a cell surface protein, then fluorescently-labeledantibodies are incubated with cells from the activation library. If thegene of interest encodes a secreted protein, then cells can bebiotinylated and incubated with streptavidin conjugated to an antibodyspecific to the protein of interest (Manz et al., Proc. Natl. Acad. Sci.(USA) 92:1921 (1995)). Following incubation, the cells are placed in ahigh concentration of gelatin (or other polymer such as agarose ormethylcellulose) to limit diffusion of the secreted protein. As proteinis secreted by the cell, it is captured by the antibody bound to thecell surface. The presence of the protein of interest is then detectedby a second antibody which is fluorescently labeled. For both secretedand membrane bound proteins, the cells can then be sorted according totheir fluorescence signal. Fluorescent cells can then be isolated,expanded, and further enriched by FACS, limiting dilution, or other cellpurification techniques known in the art.

Magnetic Bead Separation. The principle of this technique is similar toFACS. Membrane bound proteins and captured secreted proteins (asdescribed above) are detected by incubating the activation library withan antibody-conjugated magnetic beads that are specific for the proteinof interest. If the protein is present on the surface of a cell, themagnetic beads will bind to that cell. Using a magnet, the cellsexpressing the protein of interest can be purified away from the othercells in the library. The cells are then released from the beads,expanded, analyzed, and further purified if necessary.

RT-PCR. A small number of cells (equivalent to at least the number ofindividual clones in the pool) is harvested and lysed to allowpurification of the RNA. Following isolation, the RNA isreversed-transcribed using reverse transcriptase. PCR is then carriedout using primers specific for the cDNA of the gene of interest.

Alternatively, primers can be used that span the synthetic exon in theactivation construct and the exon of the endogenous gene. This primerwill not hybridize to and amplify the endogenously expressed gene ofinterest. Conversely, if the activation construct has integratedupstream of the gene of interest and activated gene expression, thenthis primer, in conjunction with a second primer specific for the genewill amplify the activated gene by virtue of the presence of thesynthetic exon spliced onto the exon from the endogenous gene. Thus,this method can be used to detect activated genes in cells that normallyexpress the gene of interest at lower than desired levels.

Phenotypic Section. In this embodiment, cells can be selected based on aphenotype conferred by the activated gene. Examples of phenotypes thatcan be selected for include proliferation, growth factor independentgrowth, colony formation, cellular differentiation (e.g.,differentiation into a neuronal cell, muscle cell, epithelial cell,etc.), anchorage independent growth, activation of cellular factors(e.g., kinases, transcription factors, nucleases, etc.), gain or loss ofcell—cell adhesion, migration, and cellular activation (e.g., restingversus activated T cells). Isolation of activated cells demonstrating aphenotype, such as those described above, is important because theactivation of an endogenous gene by the integrated construct ispresumably responsible for the observed cellular phenotype. Thus, theactivated gene may be an important therapeutic drug or drug target fortreating or inducing the observed phenotype.

The sensitivity of each of the above assays can be effectively increasedby transiently upregulating gene expression in the library cells. Thiscan be accomplished for NF-κB site-containing promoters (on theactivation construct) by adding PMA and tumor necrosis factor-α, e.g.,to the library. Separately, or in conjunction with PMA and TNF-α, sodiumbutyrate can be added to further enhance gene expression. Addition ofthese reagents can increase expression of the protein of interest,thereby allowing a lower sensitivity assay to be used to identify thegene activated cell of interest.

Since large activation libraries are created to maximize activation ofmany genes, it is advantageous to organize the library clones in pools.Each pool can consist of 1 to greater than 100,000 individual clones.Thus, in a given pool, many activated proteins are produced, often indilute concentrations (due to the overall size of the pool and thelimited number of cells within the pool that produce a given activatedprotein). Thus, concentration of the proteins prior to screeningeffectively increases the ability to detect the activated proteins inthe screening assay. One particularly useful method of concentration isultrafiltration; however, other methods can also be used. For example,proteins can be concentrated non-specifically, or semi-specifically byadsorption onto ion exchange, hydrophobic, dye, hydroxyapatite, lectin,and other suitable resins under conditions that bind most or allproteins present. The bound proteins can then be removed in a smallvolume prior to screening. It is advantageous to grow the cells in serumfree media to facilitate the concentration of proteins.

In another embodiment, a useful sequence that can be included on theactivation construct is an epitope tag. The epitope tag can consist ofan amino acid sequence that allows affinity purification of theactivated protein (e.g., on immunoaffinity or chelating matrices). Thus,by including an epitope tag on the activation construct, all of theactivated proteins from an activation library can be purified. Bypurifying the activated proteins away from other cellular and mediaproteins, screening for novel proteins and enzyme activities can befacilitated. In some instances, it may be desirable to remove theepitope tag following purification of the activated protein. This can beaccomplished by including a protease recognition sequence (e.g., FactorIIa or enterokinase cleavage site) downstream from the epitope tag onthe activation construct. Incubation of the purified, activatedprotein(s) with the appropriate protease will release the epitope tagfrom the proteins(s).

In libraries in which an epitope tag sequence is located on theactivation construct, all of the activated proteins can be purified awayfrom all other cellular and media proteins using affinity purification.This not only concentrates the activated proteins, but also purifiesthem away from other activities that can interfere with the assay usedto screen the library.

Once a pool of clones containing cells over-expressing the gene ofinterest is identified, steps can be taken to isolate the activatedcell. Isolation of the activated cell can be accomplished by a varietyof methods known in the art. Examples of cell purification methodsinclude limiting dilution, fluorescence activated cell sorting, magneticbead separation, sib selection, and single colony purification usingcloning rings.

In preferred embodiments of the invention, the methods include a processwherein the expression product is purified. In highly preferredembodiments, the cells expressing the endogenous gene product arecultured so as to produce amounts of gene product feasible forcommercial application, and especially diagnostic and therapeutic anddrug discovery uses.

Any vector used in the methods described herein can include anamplifiable marker. Thereby, amplification of both the vector and theDNA of interest (i.e., containing the over-expressed gene) occurs in thecell, and further enhanced expression of the endogenous gene isobtained. Accordingly, methods can include a step in which theendogenous gene is amplified.

Once the activated cell has been isolated, expression can be furtherincreased by amplifying the locus containing both the gene of interestand the activation construct. This can be accomplished by each of themethods described below, either separately or in combination.

Amplifiable markers are genes that can be selected for higher copynumber. Examples of amplifiable markers include dihydrofolate reductase,adenosine deaminase, aspartate transcarbamylase, dihydro-orotase, andcarbamyl phosphate synthase. For these examples, the elevated copynumber of the amplifiable marker and flanking sequences (including thegene of interest) can be selected for using a drug or toxic metabolitewhich is acted upon by the amplifiable marker. In general, as the drugor toxic metabolite concentration increases, cells containing fewercopies of the amplifiable marker die, whereas cells containing increasedcopies of the marker survive and form colonies. These colonies can beisolated, expanded, and analyzed for increased levels of production ofthe gene of interest.

Placement of an amplifiable marker on the activation construct resultsin the juxtaposition of the gene of interest and the amplifiable markerin the activated cell. Selection for activated cells containingincreased copy number of the amplifiable marker and gene of interest canbe achieved by growing the cells in the presence of increasing amountsof selective agent (usually a drug or metabolite). For example,amplification of dihydrofolate reductase (DHFR) can be selected usingmethotrexate.

As drug-resistant colonies arise at each increasing drug concentration,individual colonies can be selected and characterized for copy number ofthe amplifiable marker and gene of interest, and analyzed for expressionof the gene of interest. Individual colonies with the highest levels ofactivated gene expression can be selected for further amplification inhigher drug concentrations. At the highest drug concentrations, theclones will express greatly increased amounts of the protein ofinterest.

When amplifying DHFR, it is convenient to plate approximately 1×10⁷cells at several different concentrations of methotrexate. Usefulinitial concentrations of methotrexate range from approximately 5 nM to100 nM. However, the optimal concentration of methotrexate must bedetermined empirically for each cell line and integration site.Following growth in methotrexate containing media, colonies from thehighest concentration of methotrexate are picked and analyzed forincreased expression of the gene of interest. The clone(s) with thehighest concentration of methotrexate are then grown in higherconcentrations of methotrexate to select for further amplification ofDHFR and the gene of interest. Methotrexate concentrations in themicromolar and millimolar range can be used for clones containing thehighest degree of gene amplification.

Placement of a viral origin of replication(s) (e.g., ori P or SV40 inhuman cells, and polyoma ori in mouse cells) on the activation constructwill result in the juxtaposition of the gene of interest and the viralorigin of replication in the activated cell. The origin and flankingsequences can then be amplified by introducing the viral replicationprotein(s) in trans. For example, when ori P (the origin of replicationon Epstein-Barr virus) is utilized, EBNA-I can be expressed transientlyor stably. EBNA-1 will initiate replication from the integrated ori Plocus. The replication will extend from the origin bi-directionally. Aseach replication product is created, it too can initiate replication. Asa result, many copies of the viral origin and flanking genomic sequencesincluding the gene of interest are created. This higher copy numberallows the cells to produce larger amounts of the gene of interest.

At some frequency, the replication product will recombine to form acircular molecule containing flanking genomic sequences, including thegene of interest. Cells that contain circular molecules with the gene ofinterest can be isolated by single cell cloning and analysis by Hirtextraction and Southern blotting. Once purified, the cell containing theepisomal genomic locus at elevated copy number (typically 10-50 copies)can be propagated in culture. To achieve higher amplification, theepisome can be further boosted by including a second origin adjacent tothe first in the original construct. For example, T antigen can be usedto boost the copy number of ori P/SV40 episomes to a copy number of˜1000 (Heinzel et al., J. Virol. 62:3738 (1988)). This substantialincrease in copy number can dramatically increase protein expression.

The invention encompasses over-expression of endogenous genes both invivo and in vitro. Therefore, the cells could be used in vitro toproduce desired amounts of a gene product or could be used in vivo toprovide that gene product in the intact animal.

The invention also encompasses the proteins produced by the methodsdescribed herein. The proteins can be produced from either known, orpreviously unknown genes. Examples of known proteins that can beproduced by this method include, but are not limited to, erythropoietin,insulin, growth hormone, glucocerebrosidase, tissue plasminogenactivator, granulocyte-colony stimulating factor, granulocyte/macrophagecolony stimulating factor, interferon α, interferon β, interferon γ,interleukin-2, interleukin-6, interleukin-11, interleukin-12, TGF β,blood clotting factor V, blood clotting factor VII, blood clottingfactor VIII, blood clotting factor IX blood clotting factor X, TSH-β,bone growth factor 2, bone growth factor-7, tumor necrosis factor,alpha-1 antitrypsin, anti-thrombin III, leukemia inhibitory factor,glucagon, Protein C, protein kinase C, macrophage colony stimulatingfactor, stem cell factor, follicle stimulating hormone β, urokinase,nerve growth factors, insulin-like growth factors, insulinotropin,parathyroid hormone, lactoferrin, complement inhibitors, plateletderived growth factor, keratinocyte growth factor, neurotropin-3,thrombopoietin, chorionic gonadotropin, thrombomodulin,alphaglucosidase, epidermal growth factor, FGF, macrophage-colonystimulating factor, and cell surface receptors for each of theabove-described proteins.

Where the protein product from the activated cell is purified, anymethod of protein purification known in the art may be employed.

Isolation of Cells Containing Activated Membrane Protein-Encoding Genes

Genes that encode membrane associated proteins are particularlyinteresting from a drug development standpoint. These genes and theproteins they encode can be used, for example, to develop small moleculedrugs using combinatorial chemistry libraries and high through-putscreening assays. Alternatively, the proteins or soluble forms of theproteins (e.g., truncated proteins lacking the transmembrane region) canbe used as therapeutically active agents in humans or animals.Identification of membrane proteins can also be used to identify newligands (e.g., cytokines, growth factors, and other effect or molecules)using two hybrid approaches or affinity capture techniques. Many otheruses of membrane proteins are also possible.

Current approaches to identifying genes that encode integral membraneproteins involve isolation and sequencing of genes from cDNA libraries.Integral membrane proteins are then identified by ORF analysis usinghydrophobicity plots capable of identifying the transmembrane region ofthe protein. Unfortunately, using this approach a gene encoding anintegral membrane protein can not be identified unless the gene isexpressed in the cells used to produce the cDNA library. Furthermore,many genes are only expressed in very rare cells, during shortdevelopmental windows, and/or at very low levels. As a result, thesegenes can not be efficiently identified using the currently availableapproaches.

The present invention allows endogenous genes to be activated withoutany knowledge of the sequence, structure, function, or expressionprofile of the genes. Using the disclosed methods, genes may beactivated at the transcription level only, or at both the transcriptionand translation levels. As a result, proteins encoded by the activatedendogenous gene can be produced in cells containing the integratedvector. Furthermore, using specific vectors disclosed herein, theprotein produced from the activated endogenous gene can be modified, forexample, to include an epitope tag. Other vectors (e.g., vectors 12-17described above) may encode a signal peptide followed by an epitope tag.This vector can be used to isolate cells that have activated expressionof an integral membrane protein (see Example 5 below). This vector canalso be used to direct secretion of proteins that are not normallysecreted.

Thus, the invention also is directed to methods for identifying anendogenous gene encoding a cellular integral membrane protein or atransmembrane protein. Such methods of the invention may comprise one ormore steps. For example, one such method of the invention may comprise(a) introducing one or more vectors of the invention into a cell; (b)allowing the vector to integrate into the genome of the cell bynon-homologous recombination; (c) allowing over-expression of anendogenous gene in the cell by upregulation of the gene by thetranscriptional regulatory sequence contained on the integrated vectorconstruct; (d) screening the cell for over-expression of the endogenousgene; and (e) characterizing the activated gene to determine itsidentity as a gene encoding a cellular integral membrane protein. Inrelated embodiments, the invention provides such methods furthercomprising isolating the activated gene from the cell prior tocharacterizing the activated gene.

To identify genes that encode integral membrane proteins, vectorsintegrated into the genome of cells will comprise a regulatory sequencelinked to an exonic sequence containing a start codon, a signalsequence, and an epitope tag, followed by an unpaired splice donor site.Upon integration and activation of an endogenous gene, a chimericprotein is produced containing the signal peptide and epitope tag fromthe vector fused to the protein encoded by the downstream exons of theendogenous gene. This chimeric protein, by virtue of the presence of thevector encoded signal peptide, is directed to the secretory pathwaywhere translation of the protein is completed and the protein issecreted. If, however, the activated endogenous gene encodes an integralmembrane protein, and the transmembrane region of that gene is encodedby exons located 3′ of the vector integration site, then the chimericprotein will go to the cell surface, and the epitope tag will bedisplayed on the cell surface. Using known methods of cell isolation(for example flow cytometric sorting, magnetic bead cell sorting,immunoadsorption, or other methods that will be familiar to one ofordinary skill in the art), antibodies to the epitope tag can then beused to isolate the cells from the population that display the epitopetag and have activated an integral membrane encoding gene. These cellscan then be used to study the function of the membrane protein.Alternatively, the activated gene may then be isolated from these cellsusing any art-known method, e.g., through hybridization with a DNA probespecific to the vector-encoded exon to screen a cDNA library producedfrom these cells, or using the genetic constructs described herein.

The epitope tag encoded by the vector exon may be a short peptidecapable of binding to an antibody, a short peptide capable of binding toa substance (e.g., poly histidinet divalent metal ion supports, maltosebinding protein/maltose supports, glutathione S-transferase/glutathionesupport), or an extracellular domain (lacking a transmembrane domain)from an integral membrane protein for which an antibody or ligandexists. It will be understood, however, that other types of epitope tagsthat are familiar to one of ordinary skill in the art may be usedequivalently in accordance with the invention.

Vectors for Non-targeted Activation of Endogenous Genes

As noted above, non-targeted gene activation has a number of importantapplications, including activating endogenous genes in host cells whichprovides a powerful approach to discovering and isolating new genes andproteins, and to producing large amounts of specific proteins forcommercialization. For some applications of non-targeted geneactivation, it is desirable to create libraries of cells in which eachmember of the library contains an activation vector integrated into aunique location in the host cell genome, and in which each member of thelibrary has activated a different endogenous gene. Furthermore, it wouldbe desirable to remove cells from the library that contain an integratedvector, but fail to activate an endogenous gene. Since eukaryoticgenomes often contain large regions that lack genes, integration of anactivation vector into a region devoid of genes can occur frequently.These integrated vectors, however, fail to activate an endogenous gene,and yet are capable of conferring drug resistance on the host cells whena selectable marker (driven by a suitable promoter and followed by apolyadenylation signal) is included on the activation vector. Even moreproblematic for gene discovery applications, a transcript containingvector sequences is produced in these cells regardless of whether or nota gene has been activated. In cases where a gene has not been activated,these vector sequence-containing transcripts contain non-genic genomicDNA sequences. As a result, when isolating activated genes, one cannotisolate all RNA (or cDNA) molecules that are derived from the integratedvector (i.e. transcripts containing vector sequences), since many ofthese transcripts do not encode an endogenous gene. To overcome thesedifficulties, the present invention provides highly specific vectors andmethods that facilitate isolation of vector-activated genes.

These vectors of the invention are useful for activating expression ofendogenous genes and for isolating the mRNA and cDNA corresponding tothe activated genes. One such vector reduces the number of cells inwhich the vector integrated into the genome but failed to activateexpression from (or transcription through) an endogenous gene. Byremoving these cells, fewer library members can be created and screenedto isolate a given number of activated genes. Furthermore,vector-containing cells that fail to activate gene expression produce anRNA molecule that can interfere with isolation of bona fide activatedgenes. Thus, the vectors disclosed herein are particularly useful forproducing cells suitable for protein over-expression and/or forisolating cDNA molecules corresponding to activated genes. The secondtype of vector of the invention is useful for isolating exon I fromactivated endogenous genes. As a result, these vectors can be used toobtain full-length genes from activated RNA transcripts. Each of thefunctional vector components described herein may be used separately, orin combination with each other.

Poly(A) Trap Activation Vectors

To facilitate isolation of activated genes, the present inventionprovides novel gene activation vectors that are capable of producing adrug resistant colony, preferentially upon activation of an endogenousgene. Such vectors are referred to herein as “poly(A) trap vectors.”Examples of poly(A) trap vectors are shown in FIGS. 8A-8F. Thenucleotide sequence of one such dual poly(A) trap vector, designatedpRIG21b, is shown in FIGS. 15A-15B (SEQ ID NO:19). These vectors containa transcriptional regulatory sequence (which may be any transcriptionalregulatory sequence, including but not limited to the promoters,enhancers, and repressors described herein, and which preferably is apromoter or an enhancer, and most preferably a promoter such as a CMVimmediate early gene promoter, an SV40 T antigen promoter, atetracycline-inducible promoter, or a β-actin promoter) operably linkedto a selectable marker gene lacking a poly(A) signal. Since theselectable marker gene lacks a polyadenylation signal, its message willnot be stable, and the marker gene product will not be efficientlyproduced. However, if the activation vector integrates upstream of anendogenous gene, the selectable marker can utilize the polyadenylationsignal of the endogenous gene, thereby allowing production of theselectable marker protein in sufficient amounts to confer drugresistance. Thus, cells that integrate this activation vector generallyform a drug resistant colony only if an endogenous gene has beenactivated.

The poly(A) trap activation vectors can include any selectable orscreenable marker. Furthermore, the selectable marker can be expressedfrom any promoter that is functional in the cells used to create theintegration library. Thus, the selectable marker can be expressed byviral or non-viral promoters. Optionally, an unpaired splice donor sitemay be included in the construct, preferably 3′ of the selectable markerto allow the exon encoding the selectable marker to be spliced directlyto the exons of the endogenous gene. When a downstream transcriptionalregulatory sequence and a splice donor site is included on the vector,the inclusion of a splice donor site adjacent to the selectable markerresults in the removal of these downstream elements from the messengerRNA.

In a related embodiment, a second transcriptional regulatory sequence(which may be any transcriptional regulatory sequence, including but notlimited to the promoters, enhancers, and repressors described herein,and which preferably is a promoter or an enhancer, and most preferably apromoter) may be located downstream of, and in the same orientation as,the selectable marker. Optionally, an unpaired splice donor site may belinked to the downstream transcriptional regulatory sequence. In thisconfiguration, the poly(A) trap vector is capable of producing a messagecontaining the downstream vector-encoded exon spliced to endogenousexons. As described below, these chimeric transcripts can be translatedinto native or modified protein, depending on the nature of thevector-encoded exon.

As used herein, a “vector-encoded exon” means a region of a vectordownstream of the transcriptional regulatory sequence and between thetranscription start site and the unpaired splice donor site found on thevector. The vector-encoded exon is present at the 5′ end of thetranscript containing the endogenous gene in the fully processedmessage. Analogously, as used herein, a “vector-encoded intron” is theregion of the vector located downstream of the unpaired splice donorsite. When a linearization site is present on the vector, thevector-encoded intron is the region of the vector that is downstream ofthe vector-encoded exon between the unpaired splice donor site and thelinearization site. The vector-encoded intron is removed from theactivated gene transcript during RNA processing.

Splice Acceptor Trap (SAT) Vectors

As an alternative approach for removing cells that fail to activate anendogenous gene, the invention provides additional vectors designatedherein as “Splice Acceptor Trap” (SAT) vectors. These vectors aredesigned to splice from a vector encoded splice donor site to anendogenous splice acceptor. Furthermore, the vectors are designed toproduce a product that is toxic to the host cells (or a product that canbe selected against) if splicing does not occur. Thus, these vectorsfacilitate elimination of cells in which the vector-encoded exon failedto splice to an endogenous exon.

The splice acceptor trap vectors can contain both a positive selectablemarker and a negative selectable marker gene oriented in the samedirection on the vector. As used herein, a positive selectable marker isa gene that, upon expression, produces a protein capable of facilitatingthe isolation of cells expressing the marker. Analogously, as usedherein, a negative selectable marker is a gene that, upon expression,produces a protein capable of facilitating removal of cells expressingthe marker.

The positive selectable marker and the negative selectable marker arepreferably separated in the vector construct by an unpaired splice donorsite. In other embodiments, however, the positive selectable marker maybe fused to the negative selectable marker gene. In this configuration,an unpaired splice donor site is located between the positive andnegative selectable marker, such that the reading frame of the negativeselectable marker is preserved. The unpaired splice donor site ispreferably located at the junction of the positive and negativeselectable markers. However, the unpaired splice donor site may belocated anywhere in the fusion gene such that upon splicing to anendogenous splice acceptor site, the positive selectable marker will beexpressed in an active form and the negative selectable marker will beexpressed in an inactive form, or not at all. In this configuration, thepositive selectable marker is located upstream of the negativeselectable marker.

It will also be apparent to one of ordinary skill in view of thedescription contained herein that the positive and negative selectablemarkers on the SAT vector need not be expressed as a fusion protein. Inone embodiment, an internal ribosomal entry site (ires) is insertedbetween the positive selectable marker and the negative selectablemarker. In this configuration, the unpaired splice donor site can bepositioned between the two markers, or in the open reading frame ofeither marker gene such that, upon splicing, the positive selectablemarker will be expressed in an active form and the negative selectablemarker will be expressed in an inactive form, or not at all. In anotherembodiment, the positive selectable marker may be driven from adifferent transcriptional regulatory sequence than the negativeselectable marker. In this configuration, the unpaired splice donor siteis located in the 5′ untranslated region of the negative selectablemarker or anywhere in the open reading frame of the negative selectablemarker such that, upon splicing, the negative selectable marker will beproduced in an inactive form or not at all. Furthermore, when thepositive and negative markers are driven from different transcriptionalregulatory sequences, the positive selectable marker maybe locatedupstream or downstream of the negative selectable marker, and thepositive selectable marker may contain or lack a splice donor site atits 3′ end.

The vectors described herein may contain any positive selectable marker.Examples of positive selectable markers useful in this invention includegenes encoding neomycin (neo), hypoxanthine phosphodosyl transferase(HPRT), puromycin (pac), dihydro-oratase, glutamine synthetase (GS),histidine D (his D), carbamyl phosphate synthase (CAD), dihydrofolatereductase (DBFR), multidrug resistance 1 (mdr1), aspartatetranscarbamylase, xanthine-guanine phosphoribosyl transferase (gpt), andadenosine deaminase (ada). Alternatively, the vectors may contain ascreenable marker in place of the positive selectable marker. Screenablemarkers include any protein capable of producing a recognizablephenotype in the host cell. Examples of screenable markers included cellsurface epitopes (such as CD2) and enzymes (such as β-galactosidase).

The vectors described herein may also, or alternatively, contain anynegative selectable marker that can be selected against. Examples ofnegative selectable markers include hypoxanthine phosphoribosyltransferase (HPRT), thymidine kinase (TK), and diptheria toxin. Thenegative selectable marker can also be a screenable marker, such as acell surface protein or an enzyme. Cells expressing the negativescreenable marker may be removed by, for example, Fluorescence ActivatedCell Sorting (FACS) or magnetic bead cell sorting.

To isolate cells that have activated expression of an endogenous gene,the cells containing the integrated vector can be placed under theappropriate drug selection. Selection for the positive selectable markerand against the negative selectable marker can occur simultaneously. Inanother embodiment, selection can occur sequentially. When selectionoccurs sequentially, selection for the positive selectable marker canoccur first, followed by selection against the negative selectablemarker. Alternatively, selection against the negative selectable markercan occur first, followed by selection for the positive selectablemarker.

The positive and negative markers are expressed by a transcriptionalregulatory element located upstream of the translation start site ofeach gene. When a positive/negative marker fusion gene or an iressequence is used, a single transcriptional regulatory element drivesexpression of both markers. A poly(A) signal may be placed 3′ of eachselectable marker. If a positive/negative fusion gene is used a singlepoly(A) signal is positioned 3′ of the markers. Alternatively, a poly(A)signal may be excluded from the vector to provide additional specificityfor a gene activation event (see dual poly(A)/splice acceptor trapbelow).

Dual Poly(A)/Splice Acceptor Trap Vectors

To further reduce the number of cells that lack a gene activation event,the invention also provides vectors that confers host cell survival onlyif the vector-encoded exon has spliced to an exon from an endogenousgene and has acquired a poly(A) signal. These vectors are designatedherein as “dual poly(A)/splice acceptor trap vectors” or as “dualpoly(A)/SAT vectors.” By requiring both splicing and polyadenylation tooccur for cell survival, cells that fail to activate an endogenous geneare more efficiently eliminated from the activation library.

The dual poly(A)/splice acceptor trap vectors contain a positiveselectable marker and a negative selectable marker configured asdescribed for the SAT vectors; however, neither gene contains afunctional poly(A) signal. Thus, the positive selectable marker will notbe expressed at high levels unless splicing occurs to capture anendogenous poly(A) signal. Aside from the lack of a poly(A) signal, allother features and embodiments of this type of vector are the same asthose of the SAT vectors as described herein Examples of dualpoly(A)/SAT vectors are shown in FIGS. 9A-9F and 10A-10F. The nucleotidesequence of one such dual poly(A)/SAT vector, designated pRIG22b, isshown in FIGS. 16A-16B (SEQ ID NO:20).

Vectors for Activating Protein Expression from Endogenous Genes

In many applications of non-targeted gene activation, it is desirable toproduce protein from the activated endogenous gene. To accomplish this,a second transcriptional regulatory sequence (which may be anytranscriptional regulatory sequence, including but not limited to thepromoters, enhancers, and repressors described herein, and which ispreferably a promoter or an enhancer, and most preferably a promoter)can be placed downstream of the selectable marker(s) on any of thevectors described herein. When poly(A) trap vectors, SAT vectors, ordual poly(A) trap/SAT vectors are used, the downstream transcriptionalregulatory sequence is positioned to drive expression in the samedirection as the upstream selectable marker(s). To activate expressionof full-length protein with this type of vector, however, the vectormust integrate into the 5′ UTR of the endogenous gene to avoid crypticstart ATG codons upstream of exon I.

Alternatively, to increase the frequency of protein expression usingnon-targeted gene activation, the downstream transcriptional regulatorysequence on the vector may be operably linked to an exonic sequencefollowed by a splice donor site. In a preferred embodiment, the vectorexon lacks a start codon. This vector is particularly useful foractivating protein expression from genes that do not encode thetranslation start codon in exon I. In an alternative preferredembodiment, the vector exon contains a start codon. Additional codonscan be located between the translational start codon and the splicedonor site. For example, a partial signal secretion sequence can beencoded on the vector exon. The partial signal sequence can be any aminoacid sequence capable of complementing a partial signal sequence from anendogenous gene to produce a functional signal sequence. The partialsequence may encode between one and one hundred amino acids, and may bederived from existing genes, or may consist of novel sequences. Thus,this vectoris useful for producing and secreting protein from genes thatencode part of the endogenous signal sequence in exon I, and theremainder in subsequent exons. In another example of a vector useful foractivating a particular type of endogenous gene, a functional signalsequence can be encoded on the vector exon. This vector allows proteinto be produced and secreted from genes that encode a signal sequence inexon I. It can also be used to produce secreted forms of proteins thatare not normally secreted.

In cases where a start codon is included on the vector exon, it can beadvantageous to produce a vector in each reading frame. This is achievedby varying the number of nucleotides between the start codon and thesplice donor junction site. Together, the preferred vectorconfigurations are capable of producing protein from endogenous genes,regardless of the exonfintron structure, location of the translationstart. codon, or reading frame.

Vectors for Isolating Exon I from Activated Endogenous Genes

The non-targeted gene activation vectors described above are useful foractivating and isolating endogenous genes and for producing protein fromendogenous genes. Upon integration upstream of an endogenous gene,however each of these vectors produces a transcript that lacks exon Ifrom the endogenous gene. Since the vectors are designed to produce atranscript containing the vector encoded exon spliced to the firstsplice acceptor site downstream of the vector integration site, andsince the first exon of eukaryotic genes does not contain a spliceacceptor site, normally, the first exon of endogenous genes will not berecovered on mRNA molecules derived from non-targeted gene activation.For some genes, such as genes that contain coding information in thefirst exon, there is a need to efficiently recover the first exon of theactivated endogenous gene.

To recover the first exon of activated endogenous genes, atranscriptional regulatory sequence (which may be any transcriptionalregulatory sequence, including but not limited to the promoters,enhancers, and repressors described herein, and which is preferably apromoter or an enhancer, and most preferably a promoter) is included onthe activation vector downstream of a second transcriptional regulatorysequence (which may also be any transcriptional regulatory sequence,including but not limited to the promoters, enhancers, and repressorsdescribed herein, and which is preferably a promoter or an enhancer, andmost preferably a promoter) which drives expression of a vector encodedexon. Thus, the upstream transcriptional regulatory sequence is linkedto an unpaired splice donor site and the downstream transcriptionalregulatory sequence is not linked to a splice donor site. Bothtranscriptional regulatory sequences are oriented to drive expression inthe same direction Examples of such exon I recovery vectors are shown inFIGS. 12A-12G. The integration of this type of vector will create atleast two different types of RNA transcripts (FIG. 13). The firsttranscript is derived from the upstream transcriptional regulatorysequence and contains the vector exon spliced to exon II of anendogenous gene. The second transcript is derived from the downstreamtranscriptional regulatory sequence and contains, from 5′ to 3′, theregion between the vector and the transcription start site of the gene,exon I, exon II, and all downstream exons. Using methods describedherein, both transcripts can be recovered and analyzed, allowing thecharacterization of exon I from genes isolated by non-targeted geneactivation.

The exon located on the activation vector can encode a selectablemarker, a protein, a portion of a protein, secretion signal sequences, aportion of a signal sequence, an epitope, or nothing. When a protein isencoded by the exon, a poly(A) signal may be included downstream of thevector encoded gene. Alternatively, a poly(A) signal maybe omitted. Inanother embodiment, a positive and negative selectable marker may beoperably linked to the upstream transcriptional regulatory sequence(s).In this embodiment, the position of the unpaired splice donor siterelative to the selectable markers is described above for the SATvectors and the dual poly(A)/SAT vectors.

Gene Activation Vectors for Single-Exon and Multi-Exon Gene Trapping

As noted above, in one embodiment the poly(A) trap vectors of theinvention may contain a promoter operably linked to a selectable markerfollowed by an unpaired splice donor site. Such vectors, when integratedinto or near a gene, produce transcripts containing the selectablemarker spliced onto an endogenous gene. Since the endogenous geneencodes a poly(A) signal, the resulting mRNA is polyadenylated, therebyallowing the transcript to be translated at levels sufficient to conferdrug resistance on the cell containing the integrated vector.

While the vectors described above are capable of “trapping” endogenousgenes, the splice donor site downstream of a selectable marker cannot beused in, and in some cases can interfere with, several potentialapplications for such vectors. First, these vectors cannot be used toselectively trap single exon genes, since these genes do not contain asplice acceptor site. Second, these vectors often “trap” cryptic genes,since drug resistance relies solely on vector integration upstream of apoly (A) signal. Unfortunately, cryptic poly (A) signals exist in thegenome, leading to formation of drug resistant cells and creation ofnon-genic transcripts containing the selectable marker. These cells andtranscripts can interfere with gene discovery applications using thesevectors. Third, without novel modifications such as those describedherein (see above), these vectors are not capable of efficientlyproducing protein from the activated endogenous gene. Furthermore,protein expression from an endogenous gene can be poor even when aninternal ribosome entry site (ires) is included between the selectablemarker and the splice donor site, since translation from an ires isgenerally less efficient than translation from the first start codon atthe 5′ end of a transcript. Thus, there is a need for vectors that arecapable of more specifically trapping endogenous genes, including singleexon genes, and that are capable of efficiently expressing protein fromthe activated endogenous genes.

Thus, in additional embodiments, the present invention provides suchvectors. In one such embodiment, the vector may contain a promoteroperably linked to one or more (i.e., one, two, three, four, five, ormore) selectable markers, wherein the selectable marker is not followedby a splice donor site or a poly(A) signal (see FIGS. 17A-17G). Ingeneral, upon integration into a host cell genome, this vector will failto produce sufficient quantities of selectable marker since the markertranscript will not be polyadenylated. However, if the vector integratesin close proximity to, or into, a gene, including a single exon gene,the selectable marker will acquire a poly(A) signal from the endogenousgene, thereby stabilizing the marker transcript and conferring a drugresistant phenotype on the cell. In addition to selecting for vectorintegration into or near genes, vectors according to this aspect of theinvention can also be used to recover exon I from the activated gene, asdescribed in the section of this application entitled “Vectors forIsolating Exon I from Activated Endogenous Genes.”

In a preferred embodiment, the vector can contain a second selectablemarker upstream of the first selectable marker (see FIG. 18). Theupstream selectable marker is preferably operably linked to atranscriptional regulatory sequence, most preferably a promoter.Optionally, an unpaired splice donor site can be positioned between thetranscription start site and the translation start site of the upstreamselectable marker. Alternatively, the splice donor site may be locatedanywhere in the open reading frame of the upstream selectable marker,such that, following vector integration into a host cell genome, andupon splicing from the vector encoded splice donor site to an endogenousexon, the upstream selectable marker will be produced in an inactiveform, or not at all. By selecting for cells that produce the downstreampositive selectable marker in an active form, cells containing thevector integrated into or near a gene can be isolated. Furthermore, byselecting against cells producing the upstream selectable marker in theactive form, cells in which the vector transcript has spliced to an exonfrom a multi-exon endogenous gene can be removed. In other words, thesevectors can be used to isolate cells that contain a vector integratedinto a single exon gene or into the 3′ most exon of a multi-exon genesince, in these instances, a splice acceptor site is absent between thevector encoded splice donor site and the endogenous poly (A) signal.Thus, the majority of cells containing activated multi-exon genes willnot survive selection, and as a result, cells containing activatedsingle exon genes will be greatly enriched in the library.

In another preferred embodiment, vectors according to this aspect of theinvention may contain one or more (i.e., one, two, three, four, five, ormore, and preferably one) negative selectable marker(s) upstream of thefirst selectable marker (see FIGS. 19A and 19B). The negative selectablemarker preferably is operably linked to a promoter. Optionally, anunpaired splice donor site may be positioned between the transcriptionstart site and the translation start site of the negative selectablemarker. Alternatively, the splice donor site may be located anywhere inthe open reading frame of the negative selectable marker, such that,following vector integration into a host cell genome, and upon splicingfrom the vector encoded splice donor site to an endogenous exon, thenegative selectable marker will be produced in an inactive form, or notat all. By selecting for cells that produce the positive selectablemarker in an active form and selecting against cells producing thenegative selectable marker in the active form, these vectors can be usedto identify cells containing the vector integrated into or upstream ofan endogenous gene. Since (1) splicing to an endogenous exon and (2)acquisition of a poly (A) signal are both required for cell survival,cells containing cryptic gene trap events are reduced within thelibrary. The reason for this is that the probability of a vectorintegrating next to both a cryptic splice acceptor site and a crypticpoly (A) signal is substantially less than the probability of the vectorintegrating next to a single cryptic site. Thus, these vectors provide ahigher degree of specificity for trapping genes than previous vectors.

It will also be recognized by one of ordinary skill in view of theteachings contained herein that vectors containing positive and negativeselectable markers can be used to produce protein from the activatedendogenous gene. One vector configuration capable of directing proteinproduction consists of the splice donor site positioned in the 5′ UTR ofthe negative selectable marker. Upon splicing, a chimeric transcriptcontaining the 5′ UTR from the negative selectable marker linked to thesecond exon of an endogenous gene is produced. This vector is capable ofactivating protein production from genes that encode a translation startcodon in the second or subsequent exon. Likewise, the splice donor sitecan be placed in the open reading frame of the negative selectablemarker, in a position that does not interfere with the function of themarker unless splicing has occurred. Similar vectors containing thesplice donor site positioned in different reading frames relative to thetranslation start codon can also be used. Upon splicing to an endogenousgene, these vectors will produce a chimeric transcript containing astart codon from the negative selectable marker fused to exon II of theactivated endogenous gene. Thus, these vectors will be capable ofactivating protein expression from genes that encode a translation startcodon in exon I. Additional positive/negative selection vector designscapable of efficiently producing protein from activated endogenous genesare described below.

Any of the vectors of the invention can contain an internal ribosomeentry site (ires) 3′ of the downstream selectable marker. The iresallows translation of the endogenous gene upon vector integration intoan endogenous gene. Optionally, a translation start codon may beincluded between the selectable marker and the ires sequence. When astart codon is present, additional codons may be present on the exon.The start codon, and if present additional codons, may be present inany, and collectively all, reading frames relative to the splice donorsite. Furthermore, the codons downstream of the translation start codon,if present, may encode, for example, a signal secretion signal, apartial signal sequence, a protein (including a full-length protein, aportion of a protein, a protein motif, an epitope tag, etc.), or aspacer region.

In additional preferred embodiments, any of the vectors described hereinmay contain, upstream of the selectable marker(s), a secondtranscriptional regulatory sequence (most preferably a promoter)operably linked to a exonic region, followed by an unpaired splice donorsite. This upstream exon is particularly useful for expressing proteinfrom activated endogenous genes. The exon may lack a translation startcodon. Alternatively, the exon may contain a translation start codon.When a start codon is present, additional codons may be present on theexon. The start codon, and if present additional codons, may be presentin any, and collectively all, reading frames relative to the splicedonor site. Furthermore, the codons downstream of the translation startcodon, if present, may encode, for example, a signal secretion sequence,a partial signal sequence, a protein (including a full-length protein, aportion of a protein, a protein motif, an epitope tag, etc.), or aspacer region.

Activation Vectors Useful for Detecting Protein—protein Interactions

Genetic approaches for detecting protein—protein interactions havepreviously been described (see, e.g., U.S. Pat. Nos. 5,283,173;5,468,614; and 5,667,973, the disclosures of which are fullyincorporated herein by reference). This approach relies on cloning afirst cDNA molecule next to, and in frame with, a gene fragment encodinga DNA binding domain; and cloning a second cDNA molecule next to, and inframe with, a gene fragment encoding a transcription transactivationdomain. Each chimeric gene is expressed from a promoter region locatedupstream of the chimeric gene. To detect expression, both chimeric genesare transfected into a reporter cell. If the first chimeric proteininteracts with the second chimeric protein (via the proteins encoded bythe cloned cDNA's fused to the DNA binding and transcription activationdomains), then the DNA binding domain and the transcription activationdomain will be joined within a single protein complex. As a result, theprotein—protein interaction complex can bind to the regulatory region ofthe reporter gene and activate its expression.

A limitation of this previous approach is that it is only capable ofdetecting protein—protein interactions between genes that have beencloned as cDNA. As described herein, many genes are expressed at verylow levels, in rare cell types, or during short developmental windows;and therefore, these genes are typically absent from cDNA libraries.Furthermore, many genes are too large to be isolated efficiently asfull-length clones, thereby making it difficult to use these previousapproaches.

The present invention is capable of activating protein expression fromendogenous genes or from transfected genomic DNA. Unlike previousapproaches, virtually any gene can be efficiently expressed, regardlessof its normal expression pattern. Furthermore, since the presentinvention is also capable of modifying the protein expressed from theendogenous gene (or from the transfected genomic DNA), it is alsopossible to produce chimeric proteins for use in protein—proteininteraction assays.

To detect protein—protein interactions by the present invention, twovectors are used. The first vector, generally referred to as BD/SD(binding domain/splice donor), contains a promoter operably linked to apolynucleotide encoding a DNA binding domain and an unpaired splicedonor site. The second vector, generally referred to as AD/SD(activation domain/splice donor), contains a promoter operably linked toa polynucleotide encoding a transcription activation domain and anunpaired splice donor site. To accommodate genes that have differentreading frames, the binding domain and activation domain can be encodedin each of the three possible reading frames relative to the unpairedsplice donor site. In addition, BD/SD and AD/SD vectors can have otherfunctional elements, as described herein for other vectors, includingselectable markers and amplifiable markers. The vectors may also containselectable markers oriented in a configuration that permits selectionfor cells in which the vector has activated a gene.Multi-promoter/activation exon vectors are also useful. Several examplesof BD/SD and AD/SD vectors are illustrated in FIG. 25. An exampleillustrating detection of a protein—protein interaction using thesevectors is depicted in FIG. 26.

The DNA binding domain of the BD/SD vector may encode any protein domaincapable of binding to a specific nucleotide sequence. When atranscription activation protein is used to supply the DNA bindingdomain, the transcription activation domain is omitted from the BD/SDvector. Examples of genes encoding proteins with DNA binding domainsinclude, but are not limited to, the yeast GAL4 gene, the yeast GCN4gene, and the yeast ADR1 gene. Other genes from prokaryotic andeukaryotic sources may also be used to supply DNA binding domains.

The transcription activation domain of the AD/SD vector encodes aprotein domain capable of enhancing transcription of a reporter genewhen positioned near the promoter region of the reporter gene. When atranscription activation protein is used to supply the transcriptionactivation domain, the DNA binding domain is omitted from the AD/SDvector. Examples of genes encoding proteins with transcriptionactivation domains include, but are not limited to, the yeast GAL4 gene,the yeast GCN4 gene, and the yeast ADR1 gene. Other genes fromprokaryotic and eukaryotic sources may also be used to supplytranscription activation domains.

In the present invention, protein—protein interactions are detectedusing the BD/SD and AD/SD vectors, described above, to activateexpression of genes located in stretches of genomic DNA.

In one embodiment, the BD/SD vector is integrated randomly into thegenome of a reporter cell line. As with other vectors described herein,the BD/SD vectors are capable of activating protein expression fromgenes located downstream of the vector integration site. Since theactivation exon on the BD/SD vector encodes a DNA binding domain, theactivated endogenous protein will be produced as a fusion proteincontaining the DNA binding domain at its N-terminus. Thus, byintegrating the BD/SD vector into the genome of a host cell, a libraryof fusion proteins can be created, wherein each protein will contain aDNA binding domain at its N-terminus.

It is also recognized that the AD/SD vector can be integrated into thegenome of a reporter cell line to produce a library of cells, whereineach member of the library is expressed as a different endogenous genefused to a transcription activation domain.

Once created, the BD/SD library may be transfected with a vectorexpressing a specific gene (referred to below as gene X) fused to atranscription activation domain. This allows virtually any gene encodedin the genome to be tested for an interaction to gene X. Likewise, theAD/SD library may be transfected with a vector expressing a specificgene (e.g. gene X) fused to a DNA binding domain. This allows virtuallyany gene encoded in the genome to be tested for an interaction to geneX. It is also recognized that the specific gene may be stably expressedin the host cell prior to construction of the BD/SD or AD/SD libraries.

In an alternative embodiment, genomic DNA is cloned into the BD/SDand/or AD/SD vector(s) downstream of the DNA binding domain andactivation domain, respectively. If a gene is present and correctlyoriented in the genomic DNA, then the BD/SD vector (or the AD/SD vector)will be capable of expressing the gene as a fusion protein useful fordetecting protein—protein interactions. Like integration of BD/SD (orAD/SD) vectors in situ, any gene can be tested regardless of whether ithas been previously isolated as a cDNA molecule.

In another embodiment, a second library is created in the cells of thefirst library. For example, the AD/SD vector can be integrated intocells comprising the BD/SD library. Conversely, the BD/SD vector can beintegrated into cells comprising the AD/SD library. This allows allproteins expressed as binding domain fusion proteins to be testedagainst all activation domain fusion protein. Since the presentinvention is capable of expressing substantially all of the proteins (asfusions with the binding and activation domains) in a eukaryoticorganism, this approach, for the first time, allows all combinations ofprotein—protein interactions to be tested in a single library. To surveyall protein—protein interactions in an organism, the library within alibrary must be substantially comprehensive. For example, to detect ˜50%of protein—protein interactions in an organism containing 100,000 genes,the first library must contain at least 100,000 cells, each expressingan activated gene. Within each clone of the first library, the secondvector would then be used to create a library of at least 100,000clones, each containing an activated gene. Thus, the total library wouldcontain 100,000 clones×100,000 clones, or 10¹⁰ total clones. Thisassumes all genes are activated at equal frequencies, and that each geneactivation event results in production of a fusion protein in frame withthe activated endogenous gene. To produce libraries with greater than50% coverage of protein—protein interactions, and/or to ensure thatproteins that are activated at lower frequencies are represented, largerlibraries can be created.

It is also recognized that library vs. library screens can be created inseveral ways. First, both libraries are produced, simultaneously orsequentially, by integrating BD/SD and AD/SD vectors into the genome ofthe same reporter cells. Second, a first library is created byintegrating a BD/SD vector into the genome of a reporter cell, and asecond library is produced by transfecting the AD/SD vector containingcloned genomic DNA. It is recognized that in this approach, the AD/SDlibrary may be created first, followed by introduction of a BD/SD vectorcontaining cloned genomic DNA. It is also recognized that the firstlibrary can be created by transfecting the BD/SD vector (or AD/SDvector) containing cloned genomic DNA, followed by integrating thesecond vector into the reporter cell genome. Third, both libraries arecreated, simultaneously or sequentially, by transfecting cells with aBD/SD and AD/SD vectors, wherein each vector contains a cloned fragmentof genomic DNA. Fourth, it is recognized that when cloned genomicfragments are used in either the BD/SD vector or the AD/SD vector, acDNA library may be created in the other vector and introduced intocells. This allows all of the genes present in the cDNA library to betested for interaction with all other genes in the genome.

Since library/library screens involve the creation of large libraries ofcells, it is important to maximize the frequency of gene activation andin frame fusion protein production among the members of the library.This can be accomplished in at least two ways. First, the BD/SD andAD/SD vectors can contain selectable markers in a configuration that“traps” genes. Examples of selection trap vectors are shown in FIGS. 8,9, 10, 17, 19, 21, and 25. These vectors select for cells in which theactivation vector has transcriptionally activated a gene. Second,multiple promoter/activation exon units can be included on the BD/SD andAD/SD vectors. Each promoter/activation exon unit encodes the bindingdomain (or activation domain) in a different reading frame relative tothe unpaired splice donor site. An example of a multi-promoter/exonvector is illustrated in FIG. 23. This type of vector ensures that anygene activated at the transcription level will be produced as an inframe fusion protein from on of the promoter/activation exon units onthe vector. Third, the vectors can be introduced into the reporter cellsusing efficient transfection procedures. In this respect, insertion ofBD/SD and AD/SD vectors by retroviral integration is advantageous.

Reporter cells useful in the present invention include any cell that iscapable of properly splicing the transcripts produced by the BD/SD andAD/SD vectors. The reporter cells contain a reporter gene that isexpressed at higher levels in the presence of a protein—proteininteraction between proteins expressed from BD/SD and AD/SD vectors. Thereporter gene may be a selectable marker, such as any of the markersdescribed herein. Alternatively, the reporter gene may be a screenablemarker. Examples of useful selectable markers and screenable markers aredescribed herein.

In the reporter cells, a minimal promoter is operably linked to thereporter gene. To allow increased expression of the reporter gene in thepresence of a protein—protein interaction, a DNA binding site ispositioned in or near the minimal promoter, such that the DNA bindingsite is recognized by the protein encoded by the DNA binding domainregion of the BD/SD vector. In the absence of a protein—proteininteraction, the DNA binding domain fusion protein produced from BD/SDlacks a transcription activation domain, and therefore, can not activatetranscription from the minimal promoter of the reporter gene. If,however, the DNA binding domain fusion protein produced from BD/SDinteracts with the activation domain fusion protein produced from theAD/SD vector, then the protein complex can activate expression of thereporter gene. Increased reporter gene expression can be detected usingan assay for the screenable marker, or using drug selection for aselectable marker.

It is also recognized that other reporter systems can be used inconjunction with the present invention to detect protein—proteininteractions. Specifically, any protein that contains two separabledomains, each required to be in close proximity with the other toproduce a biochemical or structural activity, can be used in conjunctionwith the present invention.

Multi-Promoter/Activation Exon Vectors

In applications of nontargeted gene activation in which the goal is toactivate protein expression from an unknown gene, a collection ofvectors typically must be used. Thus, in an additional embodiment, theinvention provides vectors containing one or more promoter/activationexon units (see FIGS. 20A-20E).

To accommodate the variety of gene structures that exist in the genomesof eukaryotic cells, vectors according to this aspect of the inventionpreferably contain a transcriptional regulatory sequence (e.g., apromoter) operably linked to an activation exon with a differentstructure. Collectively, these activation exons are capable ofactivating protein expression from substantially all endogenous genes.For example, to activate protein expression from genes that encode atranslation start codon in exon II (or exons downstream of exon II), onevector can contain a transcriptional regulatory sequence (e.g., apromoter) operably linked to an activation exon lacking a translationstart codon. To activate protein expression from all types of genes thatencode a translation start codon in exon I, three separate vectors mustbe used, each containing a transcriptional regulatory sequence (e.g., apromoter) operably linked to a different activation exon. Eachactivation exon encodes a start codon in a different reading frame.Additional activation exon configurations are also useful. For example,to activate protein expression and secretion from genes that encode aportion of their signal secretion sequence in exon I, three separatevectors must be used, each containing a transcriptional regulatorysequence (e.g., a promoter) operably linked to a different activationexon. Each activation exon encodes a partial signal sequence in adifferent reading frame. To activate protein expression and secretionfrom genes that encode their entire signal sequence in exon I, threevectors must be used, each containing a transcriptional regulatorysequence (e.g., a promoter) operably linked to a different activationexon. Each activation exon contains an entire signal secretion sequencein a different reading frame. In addition to activating expression ofgenes that encode secreted proteins, promoter/activation exons encodingentire signal sequences will also activate expression and secretion ofproteins that are not normally secreted. This, for example, canfacilitate protein purification of proteins that are normallyintracellularly localized.

Other useful coding sequences can be included on the activation exon ofvectors according to this aspect of the invention, including but notlimited to sequences encoding proteins (including full length proteins,portions of proteins, protein motifs, and/or epitope tags). As describedherein, vectors according to this aspect of the invention can beintegrated, individually or collectively, into the genome of a host cellto produce a library of cells. Each member of the library willpotentially overexpress a different endogenous protein. Thus, thesecollections of vectors make it possible to activate all or substantiallyall of the endogenous genes in a eukaryotic host cell.

When integrating a collection of vectors into host cells, as describedabove, activation of protein expression can be achieved fromsubstantially any gene. Unfortunately, to produce protein from allendogenous genes, a large number of library members must be generated.In part, this is due to the large number of genes encoded by the hostcell. In addition, using this approach, many cells will contain a vectorintegrated into or near an endogenous gene; however, the integratedvector will contain an activation exon with a structure that isincompatible with activating protein expression from the endogenousgene. For example, the vector exon may encode a start codon in readingframe 1 (relative to the splice junction), whereas the protein encodedby the first exon downstream of the integrated vector may be in readingframe 2 (relative to the splice junction). Thus, many library memberswill contain an integrated vector that has activated transcription of anendogenous gene, but that failed to produce the protein encoded by theendogenous gene.

To decrease the number of cells that fail to activate protein expressionfollowing vector integration into or near an endogenous gene, a vectorcontaining multiple promoter/activation exons can be used. On thisvector, each promoter/activation exon unit can be capable of activatingprotein expression from an endogenous gene with a different structure.Since a single vector comprising multiple activation exons is capable ofproducing multiple transcripts, each containing a different activationexon, a single vector integrated into or near a gene can be capable ofactivating protein expression, regardless of the structure of theendogenous gene (see FIG. 21).

Multi-promoter/activation exon vectors can contain two or morepromoter/activation exons. Each promoter/activation exon unit may befollowed by an unpaired splice donor site. In one such embodiment, twopromoter/activation exons are included on the vector, wherein eachpromoter/activation exon is capable of activating protein expressionfrom a different type of endogenous gene. In a preferred embodiment, thevector may contain three promoter/activation exons, wherein each exonencodes a translation start codon in a different reading frame. Inanother preferred embodiment, the vector may contain threepromoter/activation exons, wherein each exon encodes a partial signalsecretion sequence in a different reading frame. In yet anotherpreferred embodiment, the vector may contain three promoter/activationexons, wherein each exon encodes an entire signal secretion sequence ina different reading frame. Additional embodiments include each of thevectors above containing a fourth promoter/activation exon, wherein thefourth activation exon does not encode a translation start codon.

Any number (e.g., one or more, two or more, three or more, four or more,five or more, etc.) of promoter/activation exon units may be included onthe vector. When multiple promoter/activation exons are present on asingle vector, they are preferably oriented in the same directionrelative to one another (i.e., the promoters drive expression in thesame direction).

The promoters that drive transcription of different activation exons maybe the same as one another or one or more promoters may be different.The promoters may be viral, cellular, or synthetic. The promoters may beconstitutive or inducible. Other types of promoters and regulatorysequences, recognizable to one skilled in the art or as describedherein, may also be used in preparing the vectors according to thisaspect of the invention.

Any of the vectors containing multiple promoter/activation exon unitsmay optionally include one or more selectable marker(s) and/oramplifiable marker(s). The selectable and/or amplifiable markers maycontain a poly(A) signal. Alternatively, the markers may lack a poly(A)signal. The selectable marker may be a positive or negative selectablemarker. The selectable marker may contain an unpaired splice donor siteupstream, within, or downstream of the marker. Alternatively, theselectable marker may lack an unpaired splice donor site. The selectablemarker(s) and/or amplifiable marker(s), when present, may be locatedupstream, among, or downstream of the promoter/activation exon units.The selectable and/or amplifiable marker(s) may be located on the vectorin any orientation relative to the promoter/activation exon units. Whenthe purpose of the selectable marker is to trap endogenous genes, theselectable marker is preferably oriented in the same direction as thepromoter/activation exons.

Amplifiable Markers

Any of the vectors described herein may also optionally comprise one ormore (e.g., two, three, four, five, or more) amplifiable markers.Examples of amplifiable markers include those described in detailhereinabove. Preferably, the amplifiable marker(s) are located upstreamof the positive/negative selectable marker(s). When usingpolyadenylation trap vectors, it may be advantageous to omit apolyadenylation signal from the amplifiable marker(s) to eliminate thepossibility of capturing a vector-encoded poly(A) signal derived fromvector concatemerization prior to integration.

When present, the amplifiable marker(s) may be located upstream of theactivation transcriptional regulatory sequence (i.e. the promoterresponsible for directing transcription from the vector through theendogenous gene). The amplifiable marker(s) may be present on the vectorin any orientation (i.e. the open reading frame may be present on eitherDNA strand).

It is also understood that the amplifiable marker(s) can also be thesame gene as the positive selectable marker. Examples of genes that canbe used both as positive selectable markers and amplifiable markersinclude dihydrofolate reductase, adenosine deaminase (ada),dihydro-orotase, glutamine synthase (GS), and carbamyl phosphatesynthase (CAD).

In some embodiments and for certain applications, it may be desirable toplace multiple amplifiable markers on the vector. Use of more than oneamplifiable marker allows dual selection, or alternatively sequentialselection, for each amplifiable marker. This facilitates the isolationof cells that have amplified the vector and flanking genomic locus,including the gene of interest.

Promoters

It is understood that any promoter and regulatory element may be used onthese activation vectors to drive expression of the selectable marker,amplifiable marker (if present), and/or the endogenous gene. Inadditional preferred embodiments, the promoter driving expression of theendogenous gene is a strong promoter. The CMV immediate early genepromoter, SV40 T antigen promoter, and β-actin promoter are examples ofthis type of promoter. In another preferred embodiment, an induciblepromoter is used to drive expression of the endogenous genes. Thisallows endogenous proteins to be expressed in a more controlled fashion.The Tetracycline inducible promoter, heat shock promoter, ectdysonepromoter, and metallothionein promoter are examples of this type ofpromoter. In yet another embodiment, a tissue specific promoter is usedto drive expression of endogenous genes. Examples of tissue specificpromoters include, but are not limited to, immunoglobulin promoters,casein promoter, and growth hormone promoter.

Restriction Sites

The vectors of the invention can contain one or more restriction siteslocated downstream of the unpaired splice donor site in the vector.These restriction sites can be used to linearize plasmid vectors priorto transfection. In the linear configuration, the activation vectorcontains, from 5′ to 3′ relative to the transcribed strand, a promoter,a splice donor site, and a linearization site.

A restriction site(s) may also be included in the vector intron tofacilitate removal of vector intron-containing cDNA molecules. In thisembodiment, the vector contains, from 5′ to 3′ relative to thetranscribed strand, a promoter, a splice donor site, a restriction site,and a linearization site. By including a restriction site between theunpaired splice donor site and the linearization site, unsplicedtranscripts can be removed by digestion of cDNA with the appropriaterestriction enzyme. cDNA molecules derived from gene activation haveremoved the vector intron containing the restriction site, andtherefore, will not be digested. This allows gene activated transcriptsto be preferentially enriched during amplification/cloning, and greatlyfacilitates identification and analysis of endogenous genes.

A restriction site(s) may also be included in the vector exon tofacilitate cloning of activated genes. Following gene activation, mRNAis recovered from cells and synthesized into cDNA. By digesting the cDNAwith a restriction enzyme that cuts in the vector exon, gene activatedcDNA molecules will contain an appropriate overhang at the 5′ end forsubsequent cloning into a suitable vector. This facilitates isolation ofgene activated cDNA molecules.

In one embodiment, the restriction site located in the vector exon isdifferent than the restriction site(s) located in the vector intron.This facilitates removal of cDNA molecules that contain a vector intronsince the digested cDNA fragments from vector intron containingtranscripts can be designed to have an overhang that is incompatiblewith the cloning vector (see below). Alternatively, degeneraterestriction sites recognized by the same enzyme may be located in thevector exon and intron. Enzymes that cleave these sites are capable ofcleaving multiple sites, sites with an odd number of bases in therecognition sequence, sites with interrupted palindromes, nonpalindromicsequences, or sites containing one or more degenerate bases. In otherwords, restriction sites recognized by the same restriction endonucleasemay be used if the enzyme produces an overhang in the vector exon thatis different from the overhang produced in the vector intron. Sincedifferent overhangs are produced, a cloning vector containing a sitethat is compatible with the vector exon overhang, and incompatible withthe vector intron overhang may be used to preferentially clone vectorexon containing and vector intron lacking cDNA molecules. Examples ofuseful degenerate restriction sites include DNA sequences recognized bySfi I, Acci, Afl III, SapI, Ple I, Tsp45 I, ScrF I, Tse I, PpuM I, RsrII, and SgrA I.

The restriction site(s) located in the vector intron and/or exon can bea rare restriction site (e.g. an 8 bp restriction site) or an ultra-raresite (e.g. a site recognized by intron encoded nucleases). Examples ofrestriction enzymes with 8 bp recognitions sites include NotI, SfiI,PacI, AscI, FseI, PmeI, SgfI, SrfI, SbfI, Sse 8387 I, and SwaI. Examplesof intron encoded restriction enzymes include I-PpoI, I-SceI, I-CeuI,PI-PspI, and PI-TliI. Alternatively, restriction sites smaller than 8 bpcan be placed on the vector. For example, restriction sites composed of7 bp, 6 bp, 5 bp, or 4 bp can be used. In general, the use of smallerthe restriction recognition sites will lead to the cloning of less thanfull-length genes. In some cases, such as creation of hybridizationprobes, isolation of smaller cDNA clones may be advantageous.

Bidirectional Activation Vectors

The activation vectors described herein can also be bidirectional. Whena single activation transcriptional regulatory sequence is present onthe vector, gene activation occurs only when the vector integrates intoan appropriate location (e.g. upstream of the gene) and in the correctorientation. That is, in order to activate an endogenous gene, thepromoter on the activation construct must face the endogenous geneallowing transcription of the coding strand. As a result of thisdirectionality requirement, only half of the integration events into alocus may result in the transcriptional activation of an endogenousgene. The other half of integration events result in the vectortranscribing away from a gene of interest Therefore, to increase thegene activation frequency by a factor of two, the present inventionprovides bidirectional vectors that may be used to activate anendogenous gene regardless of the orientation in which the vectorintegrates into the host cell genome.

A bidirectional vector according to this aspect of the inventionpreferably comprises two transcriptional regulatory sequences (which maybe any transcriptional regulatory sequences, including but not limitedto the promoters, enhancers, and repressors described herein, and whichpreferably are promoters or enhancers, and most preferably promoters),two splice donor sites, and a linearization site. When a splice donorsite is useful, each transcriptional regulatory sequence is operablylinked to a separate splice donor site, and the transcriptionalregulatory sequence/splice donor pairs may be in inverse orientationrelative to each other (i.e., the first transcriptional regulatorysequence may be integrated into the host cell genome in an orientationthat is inverse relative to the orientation in which the secondtranscriptional regulatory sequence has integrated into the host cellgenome). The two opposing transcriptional regulatory sequence/splicedonor sites can be separated by the linearization site. The function ofthe linearization site is to produce free DNA ends between thetranscriptional regulatory sequence/splicedonor sites (i.e. in alocation suitable for activation of endogenous genes). Examples ofbidirectional vectors of the invention are shown in FIGS. 11A-11C.

The two opposing transcriptional regulatory sequences may be the sametranscriptional regulatory sequences or different transcriptionalregulatory sequences. Optionally, a translational start codon (e.g. ATG)and one or more additional codons may be included on either or bothvector encoded exons. When a translational start codon is present,either or both vector exons may encode a protein a portion of a protein,a signal secretion sequence, a portion of a signal secretion sequence, aprotein motif, or an epitope tag. Alternatively, either or both vectorexons may lack a translational start codon.

The bidirectional vectors according to this aspect of the invention mayoptionally include one or more selectable markers and one or moreamplifiable markers, including those selectable markers and amplifiablemarkers described in detail herein. The bidirectional vectors may alsobe configured as poly(A) trap, splice acceptor trap, or dualpoly(A)/splice acceptor trap vectors, as described above. Other vectorconfigurations described for unidirectional vectors may also beincorporated into bidirectional vectors.

Co-transfection of Genomic DNA with Non-targeted Activation Vectors

It is recognized that any of the vectors described herein can beintegrated into, or otherwise combined with, genomic DNA prior totransfection into a eukaryotic host cell. This permits high levelexpression from virtually any gene in the genome, regardless of thenormal expression characteristics of the gene. Thus, the vectors of theinvention can be used to activate expression from genes encoded byisolated genomic DNA fragments. To accomplish this, the vector isintegrated into, or otherwise combined with, genomic DNA containing atleast one gene, or portion of a gene. Typically, the activation vectormust be positioned within or upstream of a gene in order to activategene expression. Once inserted (or joined), the downstream gene may beexpressed (as a transcript or a protein) by introducing thevector/genomic DNA into an appropriate eukaryotic host cell. Followingintroduction into the host cell, the vector encoded promoter drivesexpression through the gene encoded in the isolated DNA, and followingsplicing, produces a mature mRNA molecule. Using appropriate activationvectors, this process allows protein to be expressed from any geneencoded by the transfected genomic DNA. In addition, using the methodsdescribed herein, cDNA molecules, corresponding to genes encoded by thetransfected genomic DNA, can be generated and isolated.

To achieve stable expression of the activated gene, the transfectedactivation vector/genomic DNA can be integrated into the host cellgenome. Alternatively, the transfected activation vector/genomic DNA canbe maintained as a stable episome (e.g. using a viral origin ofreplication and/or nuclear retention function—see below). In yet anotherembodiment, the activated gene may be expressed transiently, forexample, from a plasmid.

As used herein, the term “genomic DNA” refers to the unspliced geneticmaterial from a cell. Splicing refers to the process of removing intronsfrom genes following transcription. Thus, genomic DNA, in contrast tomRNA and cDNA, contains exons and introns in an unspliced form In thepresent invention, genomic DNA derived from eukaryotic cells isparticularly useful since most eukaryotic genes contain exons andintrons, and since many of the vectors of the present invention aredesigned to activate genes encoded in the genomic DNA by splicing to thefirst downstream exon, and removing intervening introns.

Genomic DNA useful in the present invention may be isolated using anymethod known in the art. A number of methods for isolating highmolecular weight genomic DNA and ultra-high molecular weight genomic DNA(intact and encased in agarose plugs) have been described (Sambrook etal., Molecular Cloning, Cold Spring Harbor Laboratory Press, (1989)). Inaddition, commercial kits for isolating genomic DNA of various sizes arealso available (Gibco/BRL, Stratagene, Clontech, etc.).

The genomic DNA used in the invention may encompass the entire genome ofan organism. Alternatively, the genomic DNA may include only a portionof the entire genome from an organism. For example, the genomic DNA maycontain multiple chromosomes, a single chromosome, a portion of achromosome, a genetic locus, a single gene, or a portion of a gene.

Genomic DNA useful in the invention may be substantially intact (i.e.unfragmented) prior to introduction into a host cell. Alternatively, thegenomic DNA may be fragmented prior to introduction into a host cell.This can be accomplished by, for example, mechanical shearing, nucleasetreatment, chemical treament, irradiation, or other methods known in theart. When the genomic DNA is fragmented, the fragmentation conditionsmay be adjusted to produce DNA fragments of any desirable size.Typically, DNA fragments should be large enough to contain at least onegene, or a portion of a gene (e.g. at least one exon). The genomic DNAmay be introduced directly into an appropriate eukaryotic host cellwithout prior cloning. Alternatively, the genomic DNA (or genomic DNAfragments) may be cloned into a vector prior to transfection. Usefulvectors include, but are not limited to, high and intermediate copynumber plasmids (e.g. pUC, pBluescript, pACYC184, pBR322, etc.),cosmids, bacterial artificial chromosomes (BAC's), yeast artificialchromosomes (YAC's), P1 artificial chromosomes (PAC's), and phage (e.g.lambda, M13, etc.). Other cloning vectors known in the art may also beused. When genomic DNA has been cloned into a cloning vector, specificcloned DNA fragments may be isolated and used in the present invention.For example, YAC, BAC, PAC, or cosmid libraries can be screened byhybridization to identify clones that map to specific chromosomalregions. Optionally, once isolated, these clones can be ordered toproduce a contig through the chromosomal region of interest. To rapidlyisolate cDNA copies of the genes present in this contig, these genomicclones may be transfected, separately or en masse, with the activationvector into a host cell. cDNA containing a vector encoded exon, andlacking a vector encoded intron, can then be isolated and analyzed.Thus, since all genes present in a contig can be rapidly isolated ascDNA clones, this approach greatly enhances the speed of positionalcloning approaches.

Any activation vector described herein, including derivatives recognizedby those skilled in the art, may be co-transfected with genomic DNA, andtherefore, are useful in the present invention. In its simplest form,the vector can contain a promoter operably linked to an exon followed byan unpaired splice donor site. Examples of other useful vectors include,but are not limited to, poly A trap vectors (e.g. vectors illustrated inFIGS. 8, 9, 11C, 12F, and 17), dual poly (A)/Splice acceptor trapvectors (e.g. vectors illustrated in FIGS. 9, 10, 12G, 19, and 21),bi-directional vectors (e.g. vectors illustrated in FIG. 11), singleexon trap vectors (e.g. the vector illustrated in FIG. 19),multi-promoter/activation exon vectors (e.g. the vector illustrated inFIG. 23), vectors for isolating cDNA's corresponding to activated genes,and vectors for activating protein expression from activated genes (e.g.vectors illustrated in FIGS. 2, 3, 4, 8B-F, 9B-C, 9E-F, 10B-C, 10E-F,11, 12, 17B-G, and 23).

The activation vector may also contain a viral origin of replication.The presence of a viral origin of replication allows vectors containinggenomic fragments to be propagated as an episome in the host cell.Examples of useful viral origins of replication include ori P EpsteinBarr Virus), SV40 ori, BPV ori, and vaccinia ori. To facilitatereplication from these origins, the appropriate viral replicationproteins may be expressed from the vector. For example, EBV ori P andSV40 ori containing vectors may also encode and express EBNA-1 or Tantigen, respectively. Alternatively, the vectors may be introduced intocells that are already expressing the viral replication protein (e.g.EBNA-1 or T antigen). Examples of cells expressing EBNA-1 and T antigeninclude human 293 cells transfected with an EBNA-1 expression unit(Clontech) and COS-7 cells (American Type Culture Collection; ATCC No.CRL-1651), respectively.

The activation vector may also contain an amplifiable marker. Thisenables cells containing increased copies of the vector and flankinggenomic DNA, either episomal or integrated in the host cell genome, tobe isolated. Cells containing increased copies of the vector andflanking genomic DNA express the activated gene at higher levels,facilitating gene isolation and protein production.

The activation vector and genomic DNA may be introduced into any hostcell capable of splicing from the vector-encoded splice donor site to asplice acceptor site encoded by the genomic DNA. In a preferredembodiment, the genomic DNA/activation vector are transfected into ahost cell from the same species as the cell from which the genomic DNAwas isolated. In some instances, however, it is advantageous totransfect the genomic DNA into a host cell from a species that isdifferent from the cell from which the genomic DNA was isolated. Forexample, transfection of genomic DNA from one species into a host cellof a second species can facilitate analysis of the genes activated inthe transfected genomic DNA using hybridization techniques. Under highstringency hybridization, activated genes that were encoded by thetransfected DNA can be distinguished from genes derived from the hostcell. Transfection of genomic DNA from one species into a host cell fromanother species can also be used to produce protein in a heterologouscell. This may allow protein to be produced in heterologous cells thatprovide growth, protein modification, or manufacturing advantages.

The activation vector may be co-transfected into a host cell along withgenomic DNA, wherein the vector is not attached to the genomic DNA priorto introduction into the cell. In this embodiment, the genomic DNA willbecome fragmented during the transfection process, thereby creating freeDNA ends. These DNA ends can become joined to the co-transfectedactivation vector by the cell's DNA repair machinery. Following joiningto the activation vector, the genomic DNA and activation vector can beintegrated into the host cell genome by the process of non-homologousrecombination. If, during this process, a vector becomes joined to agene encoded by the transfected genomic DNA, the vector will activateits expression.

Alternatively, the non-targeted activation vector may be physicallylinked to the genomic DNA prior to transfection. In a preferredembodiment, genomic DNA fragments are ligated to the vector prior totransfection. This is advantageous because it maximizes the probabilityof the vector becoming operably linked to a gene encoded by the genomicDNA, and minimizes the probability of the vector integrating into thehost cell genome without the heterologous genomic DNA.

In a related embodiment, the genomic DNA may be cloned into theactivation vector, downstream of the activation exon. In thisembodiment, cloning of large genomic fragments can be facilitated invectors capable of accommodating large genomic fragments. Thus, theactivation vector may be constructed in BAC's, YAC's, PAC's, cosmids, orsimilar vectors capable of propagating large fragments of genomic DNA.

Another method for joining the activation vector to genomic DNA involvestransposition. In this embodiment, the activation vector is integratedinto the genomic DNA by transposition or retroviral integrationreactions prior to transfection into a cell. Accordingly, activationvectors can contain cis sequences necessary for facilitatingtransposition and/or retroviral integration. Examples of vectorscontaining transposon signals are illustrated in FIG. 27; however, it isrecognized that any vector described herein may contain transposonsignals.

Any transposition system capable of inserting foreign sequences intogenomic DNA can be used in the present invention In addition,transposons capable of facilitating inversions and deletions can also beused to practice the invention. While deletion and inversion systems donot integrate the activation vector into genomic DNA, they do allow theactivation vector to change positions relative to cloned genomic DNAwhen the genomic DNA has been cloned into the activation vector. Thus,multiple genes within a given genomic fragment can be activated byshuffling the activation vector (by integration, inversion, or deletion)into multiple positions within, or outside of, the genomic fragment.Examples of transposition systems useful for the present inventioninclude, but are not limited to δγ, Tn3, Tn5, Tn7, Tn9, Tn10, Ty,retroviral integration and retro-transposons (Berg et al., Mobile DNA,ASM Press, Washington D.C., pp. 879-925 (1989); Strathman et al., Proc.Natl. Acad Sci. USA 88:1247 (1991); Berg et al., Gene 113:9 (1992); Liuet al., Nucl. Acids Res. 15:9461 (1987), Martin et al., Proc. Natl. AcadSci. USA 92:8398 (1995); Phadnis et al., Proc. Natl. Acad Sci. USA86:5908 (1989); Tomcsanyi et al., J. Bacteriol. 172:6348 (1990); Way etal., Gene 32:369 (1984); Bainton et al., Cell 65:805 (1991); Ahmed etal., J. Mol. Biol. 178:941 (1984); Benjamin et al., Cell 59:373 (1989);Brown et al., Cell 49:347 (1987); Bichinger et al., Cell 54:955 (1988);Eichinger et al., Genes Dev. 4:324 (1990); Braitermanet al., Mol. Cell.Biol. 14:5719 (1994); Braiterman et al., Mol. Cell. Biol. 14:5731(1994); York et al., Nucl. Acids Res. 26:1927(1998); Devine et al.,Nucl. Acids Res. 18:3765 (1994); Goryshin et al., J. Biol. Chem.273:7367 (1998).

Using transposition, an activation vector may be integrated into anyform of genomic DNA. For example, the activation vector maybe integratedinto either intact or fragmented genomic DNA. Alternatively, theactivation vector may be integrated into a cloned fragment of genomicDNA (FIG. 28). In this embodiment, the genomic DNA may reside in anycloning vector, including high and intermediate copy number plasmids(e.g. pUC, pBluescript, pACYC184, pBR322, etc.), cosmids, bacterialartificial chromosomes (BAC's), yeast artificial chromosomes (YAC's), P1artificial chromosomes (PAC's), and phage (e.g. lambda, M13, etc.).Other cloning vectors known in the art may also be used. As describedabove, genomic fragments from specific genetic loci may be isolated anused as a substrate for activation vector integration.

Following integration of the activation vector, the genomic DNA may beintroduced directly into a suitable host cell for expression of theactivated gene. Alternatively, the genomic DNA may be introduced intoand propagated in an intermediate host cell. For example, followingintegration of an activation vector into a BAC genomic library, the BAClibrary can be transformed into E. coli. This allows plasmids containingthe transposon to be enriched by selecting for an antibiotic resistancemarker residing on the activation vector. As a result, BAC plasmidslacking an integrated activation vector will be removed by antibioticselection.

The transposition mediated activation vector integration may occur invitro using purified enzymes. Alternatively, the transposition reactionmay occur in vivo. For example, transposition may be carried out inbacteria, using a donor strain carrying the transposon either on avector or as integrated copies in the genome. A target of interest isintroduced into the transposer host where it receives integrations.Targets bearing insertions are then recovered from the host by geneticselection. Similarly, eukaryotic host cells, such as yeast, plant,insect, or mammalian cells, can be used to carry out the transposonmediated integration of an activation vector into a fragment of genomicDNA.

Isolation of mRNA and cDNA Produced from Activated Endogenous Genes

In additional embodiments, the present invention is directed to methodsfor isolating genes, particularly genes contained within the genome of aeukaryotic cell, that are activated using the vectors of the invention.These methods exploit the structure of the mRNA molecules produced usingthe non-targeted gene activation vectors of the invention. The methodsof the invention described herein allow virtually any activated gene tobe isolated, regardless of whether it has been previously isolated andcharacterized, and regardless of whether it has a known biologicalactivity. This is made possible by the nature of the chimerictranscripts produced from the integrated vectors of the presentinvention. Using methods described herein, activation vectors can beintegrated into the genome of a cell. Typically, the activation vectors,however, are integrated into the genome of many cells to produce alibrary of unique integration events. Each member of the librarycontains the vector located at a unique integration site(s), andpotentially contains an activated endogenous gene. Gene activationoccurs when the activation vector integrates upstream of the 3′-mostexon of an endogenous gene and in an orientation capable of allowingtranscription from the vector to proceed through the endogenous gene.The integration site may be in an intron or exon of the endogenous gene,or may be upstream of the transcription start site of the gene.Following integration, the activation constructs are designed to producea transcript capable of splicing from an exon encoded by the activationvector to an exon encoded by the endogenous gene. As a result, achimeric message is produced that contains the vector exon linked to theexons from an endogenous gene, wherein the endogenous exons are derivedfrom the region located downstream of the vector integration site. Thestructure of this chimeric transcript can be exploited for genediscovery purposes. For example, the chimeric transcripts can be rapidlyisolated to use as probes (to isolate the full length cDNA or genomiccopy of the gene or to characterize the gene) or for direct sequencingand/or characterization.

To isolate the chimeric transcripts activated by vector insertion, cDNAis produced from a library member containing the activation event. It isalso possible to isolate chimeric transcripts from pools of librarymembers in order to increase the through-put of the procedure. cDNA canthen be produced from the mRNA harvested from the activated cells.Alternatively, total RNA may be used to produce cDNA. In either case,first strand synthesis can be carried out using an oligo dT primer, anoligo dT/poly(A) signal primer, or a random primer. To facilitatecloning of the cDNA product, a poly dT based primer can be used with thestructure: 5′-Primer X(dT)₁₋₁₀₀₋3′. The oligo dT/poly(A) signal primercan have the structure 5′-(dT)₁₀₋₃₀-Primer X-N₀₋₆-TTTATT-3′. The randomprimer can have the structure: 5′-(Primer X)NNNNNN-3′. In each primer,Primer X is any sequence that can be used to subsequently PCR amplifytarget nucleic acid molecules. Where the activated gene amplificationproduct is to be cloned, it is useful to include one or more restrictionsites within the primer X sequence to facilitate subsequent cloning.Other primers recognized by those skilled in the art can be used tocreate first strand cDNA products, including primers that lack a PrimerX region.

In accordance with the invention, the primers may be conjugated with oneor more hapten molecules to facilitate subsequent isolation of nucleicacid molecules (e.g., first and/or second strand cDNA products)comprising such primers. After the primer becomes associated with thenucleic acid molecule (via incorporation during cDNA synthesis),selective isolation of the molecule containing the haptenylated primermay be accomplished using a corresponding ligand which specificallyinteracts with and binds to the hapten via ligand-hapten interactions.In preferred such aspects, the ligand may be bound to, for example, asolid support. Once bound to the solid support, the molecules ofinterest (haptenylated primer-containing nucleic acid molecules) can beseparated from contaminating nucleic acids and other materials bywashing the support matrix with a solution, preferably a buffer orwater. Cleavage of one or more of the cleavage sites within the primer,or by treatment of the solid support containing the nucleic acidmolecule with a high ionic strength elution buffer, then allows forremoval of the nucleic acid molecule of interest from the solid support.

Preferred solid supports for use in this aspect of the inventioninclude, but are not limited to, nitrocellulose, diazocellulose, glass,polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran,Sepharose, agar, starch, nylon, latexbeads, magneticbeads,paramagneticbeads, super paramagnetic beads or microtitre plates andmost preferably a magnetic bead, a paramagnetic bead or asuperparamagnetic bead, that comprises one or more ligand moleculesspecifically recognizing and binding to the hapten molecule on theprimer.

Particularly preferred hapten molecules for use on the primer moleculesof the invention, include without limitation: (i) biotin; (ii) anantibody; (iii) an enzyme; (iv) lipopolysaccharide; (v) apotransferrin;(vi) ferrotransferrin; (vii) insulin; (viii) cytokines (growth factors,interleukins or colony-stimulating factors); (ix) gp120; (x) β-actin;(xi) LFA-1; (xii) Mac-1; (xiii) glycophorin; (xiv) laminin; (xv)collagen; (xvi) fibronectin; (xvii) vitronectin; (xviii) integrinsα_(v)β₁ and α_(v)β₃; (xix) integrins α₃β₁, α₄β₁, α₄β₇, α₅β₁, α_(v)β₁,α_(IIb)β₃, α_(v)β₃ and α_(v)β₆; (xx) integrins α₁β₁, α₂β₁, α₃β₁ andα_(v)β₃; (xxi) integrins α₁β₁, α₂β₁, α₃β₁, α₆β₁, α₇β₁ and α₆β₅; (xxii)ankyrin; (xxiii) C3bi, fibrinogen or Factor X; (xxiv) ICAM-1 or ICAM-2;(xxv) spectrin or fodrin; (xxvi) CD4; (xxvii) a cytokine (e.g., growthfactor, interleukin or colony-stimulating factor) receptor; (xxviii) aninsulin receptor; (xxix) a transferrin receptor; (xxx) Fe⁺⁺⁺; (xxxi)polymyxin B or endotoxin-neutralizing protein (ENP); (xxxii) anenzyme-specific substrate; (xxxiii) protein A, protein G, a cell-surfaceFc receptor or an antibody-specific antigen; and (xxxiv) avidin andstreptavidin. Particularly preferred is biotin.

Particularly preferred ligand molecules according to this aspect of theinvention, which correspond in order to the above-described haptenmolecules, include without lirnitation: (i) avidin and streptavidin;(ii) protein A, protein G, a cell-surface Fc receptor or anantibody-specific antigen; (iii) an enzyme-specific substrate; (iv)polymyxin B or endotoxin-neutralizing protein (ENP); (v) Fe⁺⁺⁺; (vi) atransferrin receptor, (vii) an insulin receptor; (viii) a cytokine(e.g., growth factor, interleukin or colony-simulating factor) receptor;(ix) CD4; (x) spectrin or fodrin; (xi) ICAM-1 or ICAM-2; (xii) C3bifibrinogen or Factor X; (xiii) ankyrin; (xiv) integrins α₁β₁, α₂β₁,α₃β₁, α₆β₁, α₇β₁ and α₆β₅; (xv) integrins α₁β₁, α₂β₁, α₃β₁ and α_(v)β₃;(xvi) integrins α₃β₁, α₄β₁, α₄β₇, α₅β₁, α_(v)β₁, α_(IIb)β₃, α_(v)β₃ andα_(v)β₆; (xvii) integrins α_(v)β₁ and α_(v)β₃; (xviii) vitronectin;(xix) fibronectin; (xx) collagen; (xxi) laminin; (xxii) glycophorin;(xxiii) Mac-1; (xxiv) LFA-1; (xxv) β-actin; (xxvi) gp120; (xxvii)cytokines (growth factors, interleukins or colony-stimulating factors);(xxviii) insulin; (xxix) ferrotransferrin; (xxx) apotransferrin; (xxxi)lipopolysaccharide; (xxxii) an enzyme; (xxxiii) an antibody; and (xxxiv)biotin. Particularly preferred, for use with biotinylated primers of theinvention, are avidin and streptavidin.

Following first strand synthesis, second strand cDNA synthesis may becarried out using a primer specific for the vector encoded exon. Thiscreates double stranded cDNA from all transcripts that were derived fromthe vector encoded promoter. All cellular mRNA (and cDNA) produced fromendogenous promoters remains single stranded since the transcript lacksa vector exon at it 5′ end. Once second strand synthesis is carried out,the cDNA may be digested with a restriction enzyme, cloned into avector, and propagated.

To facilitate cloning, cDNA molecules containing the vector exon areamplified by PCR using a primer specific for the vector exon and aprimer specific for the first strand cDNA primer (e.g. Primer X). PCRamplification results in the production of variable length DNA fragmentsrepresenting different locations of priming during first strandsynthesis and/or amplification of multiple chimeric transcripts fromdifferent genes. These amplification products can be cloned intoplasmids for characterization, or can be labeled and used as a probe.

Other amplification techniques, such as linear amplification using RNApolymerase (Van Gelder, Proc. Natl. Acad. Sci. USA 87:1663-1667 (1990);Eberwine, Methods 10:283-288 (1996)), can be used. For example, whenlinear amplification by RNA polymerase is used, a promoter (e.g. T7promoter) can be placed on the vector exon. As a result, gene activatedtranscripts will contain the promoter sequence at the 5′ end of thetranscript. Alternatively, a promoter can be ligated onto the cDNAmolecule following first strand and second strand synthesis. Usingeither strategy, RNA polymerase is then incubated with cDNA in thepresence of ribonucleotide triphosphates to create RNA transcripts fromthe cDNA. These transcripts are then reverse transcribed to producecDNA. Since RNA polymerase can create several thousand transcripts froma single cDNA molecule, and since each of these transcripts can bereverse transcribed into cDNA, a large amplification can be achieved. Aswith PCR, amplification with RNA polymerase can facilitate cloning ofactivated genes. Other types of amplification strategies are alsopossible.

In another embodiment, the vector exon containing cDNA molecules areisolated without amplification. This may be useful in instances wherebiases occur during amplification (for example, when one DNA fragmentamplifies more efficiently than another). To produce cDNA enriched fortagged messages, RNA is isolated from the activation library. A primer(e.g. a random hexamer, oligo(dT), or hybrid primers containing a primerlinked to poly(dT) or a random nucleotides) is annealed to the RNA andused to direct first strand synthesis. The first strand cDNA moleculesare then hybridized to a primer specific for the vector encoded exon.This primer directs second strand synthesis. Following second strandsynthesis, the cDNA may be digested with restriction enzymes that cut inthe vector exon and in the first strand primer (e.g. in Primer X—seeabove). The second strand products may then be cloned into a usefulvector to allow them to be propagated.

It will be apparent to one of ordinary skill in view of the descriptioncontained herein that the cDNA products made according to the methods ofthe invention may also be cloned into a cloning vector suitable fortransfection or transformation of a variety of prokaryotic (bacterial)or eukaryotic (yeast, plant or animal including human and othermammalian) cells. Such cloning vectors, which may be expression vectors,include but are not limited to chromosomal-, episomal- and virus-derivedvectors, e.g., vectors derived from bacterial plasmids orbacteriophages, and vectors derived from combinations thereof, such ascosmids and phagemids, BACs, MACs, YACs, and the like. Other vectorssuitable for use in accordance with this aspect of the invention, andmethods for insertion of DNA fragments therein and transformation ofhost cells with such cloning vectors, will be familiar to those ofordinary skill in the art.

Removal of Unspliced Transcription Products

In some instances, the activation vector will integrate into the genomein a region lacking genes. Alternatively, it may integrate into a regioncontaining a gene(s), but be oriented in a manner that results in thetranscription of the non-coding strand. In each of these instances, geneactivated transcripts are produced that contain normally untranscribedDNA sequences next to the vector encoded exon. These sequences wouldcomplicate identification and analysis of novel genes. Therefore, itwould be advantageous to selectively remove these genomic molecules.

To remove cDNA molecules that contain a vector encoded intron, thedouble strand cDNA is treated with a restriction enzyme that recognizesa sequence located in the vector encoded intron. Preferably, therestriction enzyme creates an overhang that is different from theoverhang produced by cleavage of the vector exon. This ensures thecloning of only activated genes by preventing the cleavage products fromligating into the cloning vector.

Recovery of Exon I from activated endogenous genes

To recover exon I from activated genes, specialized vectors can be usedto create non-targeted gene activation libraries. In its simplest form,this vector contains, from 5′ to 3′, a promoter, an unpaired splicedonor site, and a second promoter. The downstream promoter is orientedin the same direction as the upstream promoter. Upon integrationupstream of an endogenous gene, this type of vector produces two typesof transcripts. The first transcript contains the vector exon joined toexon II of the endogenous gene. Methods for isolating this transcriptare described above. The second transcript contains the upstream regionof the endogenous gene followed by exon I joined to exon II and otherdownstream exons from the endogenous gene FIG. 6).

Using a two step process, exon I can be recovered from cells containingthe integrated vector. First, vector exon containing transcripts (i.e.Transcript type #1, FIG. 13) are isolated using the methods describedabove. Once isolated, the 5′ end of the transcript including exon II canbe sequenced to determine the sequence of the flanking endogenous exons.Second, once the sequence of the flanking endogenous exons is known, PCRprimers capable of annealing to exon II (or a downstream exon) of theactivated gene can be developed. These primers can be used to amplifyexon I from Transcript #2 (FIG. 13) using a modified form of inverse PCR(Zeiner, M., Biotechniques 17(6): 1051-1053 (1994)). Briefly,amplification of exon I from the endogenous gene is achieved by carryingout first strand cDNA synthesis with a gene specific primer, based onthe sequence information determined above. Second strand synthesis canbe carried out using E. coli DNA polymerase I under conditions wellknown to those skilled in the art. The double strand cDNA is thendigested with a restriction enzyme that cleaves at least once in theendogenous gene upstream of the first strand cDNA primer, and that doesnot cleave in the vector exon. Following digestion, the cDNA is selfligated to produce circular molecules. Using inverted PCR primers thatanneal in the endogenous gene upstream of therestriction/circularization site, amplification by PCR produces a DNAproduct containing exon I sequences from the endogenous gene.

Method for Selecting Cells Containing Higher Levels of Gene ActivatedTranscripts/Protein

In several embodiments of the disclosed invention, the activation vectorcontains an amplifiable marker (e.g. DHFR) and a viral origin ofreplication (e.g. EBV ori P). In other embodiments, an amplifiablemarker and viral origin of replication are present on a cloning vectorcontaining a cloned fragment of genomic DNA. In yet another embodiment,the activation vector contains one element (e.g. DHFR) and a cloningvector carrying a genomic insert contains the other element (e.g. OriP). Regardless of the initial location of the amplifiable marker andviral origin, the elements are combined on the same DNA molecule priorto or during introduction into a host cell.

In addition to the cis-acting elements, a trans-acting viral protein isgenerally required for efficient replication of the episomes. Examplesof trans-acting viral proteins include EBNA-1 and SV40 T antigen. Topromote efficient replication of episomes, the trans-acting viralprotein can be expressed from the episome. Thus, the viral trans-actingprotein may be expressed from the transposing activation vector, or maybe positioned on the backbone of the cloning vector. Alternatively, thetrans-acting viral protein may be expressed by the eukaryotic host cellsinto which the episome is introduced.

Once the amplifiable marker and viral origin of replication are on thesame molecule and present in a host cell expressing the appropriateviral replication protein(s), the copy number of the episome can beincreased. To increase the copy number of the episome, the cells can beplaced under the appropriate selection. For example, if DHFR is presenton the episome, methotrexate may be added to the culture. The selectiveagent may be applied at relatively high concentrations to isolate cellsin the population that already have a high episome copy number.Alternatively, the selective agent may be applied at lowerconcentrations, and periodically increased in concentration. Two-foldincreases in drug concentration will result in step-wise increases incopy number.

To reduce the frequency of non-specific drug resistance (i.e. drugresistance that is not associated with increased copy number of theepisome), more than one amplifiable marker can be placed on the vector.Inclusion of multiple amplifiable markers on the episome allows cells tobe selected with multiple drugs (either simultaneously or sequentially).Since non-specific drug resistance is a relatively rare event, theprobability of a cell developing non-specific drug resistance tomultiple drugs is exceedingly rare. Thus, the presence of multipleamplifiable markers on the episome facilitates isolation of cells thathave a high episome copy number.

Amplification of episome copy number increases the number of transcriptsderived from the vector activated gene. This, in turn, facilitatesisolation of cDNA molecules derived from the activated gene.Furthermore, amplification of episome copy number can dramaticallyincrease protein expression from the activated gene. Higher levels ofprotein production facilitate generation of proteins for bioassayscreening, cell assay screening, and manufacturing purposes.

As a result of the highly desirable characteristics described above,vectors containing a viral origin of replication and an amplifiablemarker, and the use of these vectors to rapidly amplify the copy numberof episomal vectors, represent a break through that extends beyond thescope of activating expression of genes present in genomic DNA. Forexample, these vectors can be used to over-express cDNA encoded genes toproduce high levels of protein expression without the need to integratethe gene into a host cell genome with an amplifiable marker.Furthermore, like amplification of chromosomal sequences, cellpossessing several hundred to several thousand episomal copies of thevector can be isolated and maintained in culture. Thus, the vectorsdescribed herein, and their uses, allow high levels of cloned genomicDNA to be propagated in mammalian cells, facilitate isolation of cDNAcopies of genes present on the vector as genomic inserts, and maximizeprotein production from cloned cDNA and genomic copies of eukaryoticgenes.

Other suitable modifications and adaptations to the methods andapplications described herein will be readily apparent to one ofordinary skill in the relevant arts and may be made without departingfrom the scope of the invention or any embodiment thereof Having nowdescribed the present invention in detail, the same will be more clearlyunderstood by reference to the following examples, which are includedherewith for purposes of illustration only and are not intended to belimiting of the invention.

EXAMPLES Example 1 Transfection of Cells for Activation of EndogenousGene Expression

Method: Construction of pRIG-1

Human DHFR was amplified by PCR from cDNA produced from HT1080 cells byPCR using the primers DHFR-F1

(5′ TCCTTCGAAGCTTGTCATGGTTGGTTCGCTAAACTGCAT 3′) (SEQ ID NO:1) andDHFR-R1 (5′ AAACTTAAGATCGATTAATCATTCTTCTCATATACTTCAA 3′) (SEQ ID NO:2),and cloned into the T site in pTARGET™ (Promega) to create pTARGET:DHFRThe RSV promoter was isolated from PREP9 by digestion with NheI and XbaIand inserted into the NheI site of pTARGET:DHFR to create pTgT:RSV+DHFR.Oligonucleotides JH169 (5′ ATCCACCATGGCTACAGGTGAGTACTCG 3′) (SEQ IDNO:3) and JH170 (5′ GATCCGAGTACTCACCTGTAGCCATGGTGGATTTAA 3′) (SEQ IDNO:4) were annealed and inserted into the I-Ppo-I and NheI sites ofpTgT:RSV+DHFR to create pTgT:RSV+DHFR+Ex1. A 279 bp region correspondingto nucleotides 230-508 of pBR322 was PCR amplified using primers Tet F1(5′ GGCGAGATCTAGCGCTATATGCGTTGATGCAAT 3′) (SEQ ID NO:5) and Tet F2 (5′GGCCAGATCTGCTACCTTAAGAGAGCCGAAACAAGCGCTCATGAGCCCGAA 3′) (SEQ ID NO:6).Amplification products were digested with BglII and cloned into theBamHI site of pTgT:RSV+RSV+DHFR+Ex1 to create pRIG-1.

Transfection—Creation of pR1G-1 Gene Activation Library in HT1080 Cells

To activate gene expression, a suitable activation construct is selectedfrom the group of constructs described above. The selected activationconstruct is then introduced into cells by any transfection method knownin the art. Examples of transfection methods include electroporation,lipofection, calcium phosphate precipitation, DEAE dextran, and receptormediated endocytosis. Following introduction into the cells, the DNA isallowed to integrate into the host cell's genome via non-homologousrecombination. Integration can occur at spontaneous chromosome breaks orat artificially induced chromosomal breaks.

Method: Transfection of human cells with pRIG1. 2×10⁹ HH1 cells, anHPRT⁻ subclone of HT1080 cells, was grown in 150 mm tissue cultureplates to 90% confluency. Media was removed from the cells and saved asconditioned media (see below). Cells were removed from the plate bybrief incubation with trypsin, added to media/10% fetal bovine serum toneutralize the trypsin, and pelleted at 1000 rpm in a Jouan centrifugefor 5 minutes. Cells were washed in 1×PBS, counted, and repelleted asabove. The cell pellet was resuspended at 2.5×10⁷ cells/ml final in1×PBS (Gibco BRL Cat #14200-075). Cells were then exposed to 50 rads ofγ irradiation from a ¹³⁷Cs source. pRIG1 (FIGS. 14A-14B; SEQ ID NO:18)was linearized with BamHI, purified with phenol/chloroform, precipitatedwith ethanol, and resuspended in PBS. Purified and linearized activationconstruct was added to the cell suspension to produce a finalconcentration of 40 μg/ml. The DNA/irradiated cell mixture was thenmixed and 400 μl was placed into each 0.4 cm electroporation cuvettes(Biorad). The cuvettes were pulsed at 250 Volts, 600 μFarads, 50 Ohmsusing an electroporation apparatus (Biorad). Following the electricpulse, the cells were incubated at room temperature for 10 minutes, andthen placed into αMEM/10% FBS containing penicillin/streptomycin(Gibco/BRL). The cells were then plated at approximately 7×10⁶ cells/150mm plate containing 35 ml αMEM/10% FBS/penstrep (33% conditionedmedia/67% fresh media). Following a 24 hour incubation at 37° C., G418(Gibco/BRL) was added to each plate to a final concentration of 500μg/ml from a 60 mg/ml stock. After 4 days of selection, the media wasreplaced with fresh αMEM/10% FBS/penstrep/500 μg/ml G418. The cells werethen incubated for another 7-10 days and the culture supernatant assayedfor the presence of new protein factors or stored at −80° C. for lateranalysis. The drug resistant clones can be stored in liquid nitrogen forlater analysis.

Example 2 Use of Ionizing Irradiation to Increase the Frequency andRandomness of DNA Integration

Method: HH1 cells were harvested at 90% confluency, washed in 1×PBS, andresuspended at a cell concentration of 7.5×10⁶ cells/ml in 1×PBS. 15 μglinearized DNA (pRIG-1) was added to the cells and mixed. 400 μl wasadded to each electroporation cuvette and pulsed at 250 Volts, 600μFarads, 50 Ohms using an electroporation apparatus (Biorad). Followingthe electric pulse, the cells were incubated at room temperature for 10minutes, and then placed into 2.5 ml αMEM/10% FBS/1×penstrep. 300 μl ofcells from each shock were irradiated at 0, 50, 500, and 5000 radsimmediately prior to or at either 1 hour or 4 hours post transfection.Immediately following irradiation, the cells were plated onto tissueculture plates in complete medium. At 24 hours post plating, G418 wasadded to the culture to a final concentration of 500 μg/ml. At 7 dayspost-selection, the culture medium was replaced with fresh completemedium containing 500 μg/ml G418. At 10 days post selection, medium wasremoved from the plate, the colonies were stained with CoomassieBlue/90% methanol/10% acetic acid and colonies with greater than 50cells were counted.

Example 3 Use of Restriction Enzymes to Generate Random, Semi-random, orTargeted Breaks in the Genome

Method: HHI cells were harvested at 90% confluence, washed in 1×PBS, andresuspended at a cell concentration of 7.5×10⁶ cells/ml in 1×PBS. Totest the efficiency of integration, 15 μg linearized DNA (PGK-βgeo) wasadded to each 400 μl aliquot of cells and mixed. To several aliquots ofcells, restriction enzymes XbaI, NotI, HindIII IppoI (10-500 units) werethen added to separate cel/DNA mixture. 400 μl was added to eachelectroporation cuvette and pulsed at 250 Volts, 600 μFarads, 50 Ohmsusing an electroporation apparatus (BioRad). Following the electricpulse, the cells were incubated at room temperature for 10 minutes, andthen placed into 2.5 ml αMEM10% FBS/1×penstrep. 300 μl of 2.5 ml totalcells from each shock were plated onto tissue culture plates in completemedia. At 24 hours post plating, G418 was added to the culture to afinal concentration of 600 μg/ml. At 7 days post-selection, the mediawas replaced with fresh complete media containing 600 μg/ml G418. At 10days post selection, media was removed from the plate, the colonies werestained with Coomassie Blue/90% methanol/10% acetic acid and colonieswith greater than 50 cells were counted.

Example 4 Amplification by Selecting for Two Amplifiable Markers Locatedon the Integrated Vector

Following integration of the vector into the genome of a host cell, thegenetic locus may be amplified in copy number by simultaneous orsequential selection for one or more amplifiable markers located on theintegrated vector. For example, a vector comprising two amplifiablemarkers may be integrated into the genome, and expression of a givengene (i. e., a gene located at the site of vector integration) can beincreased by selecting for both amplifiable markers located on thevector. This approach greatly facilitates the isolation of clones ofcells that have amplified the correct locus (i.e., the locus containingthe integrated vector).

Once the vector has been integrated into the genome by nonhomologousrecombination, individual clones of cells containing the vectorintegrated in a unique location may be isolated from other cellscontaining the vector integrated at other locations in the genome.Alternatively, mixed populations of cells may be selected foramplification.

Cells containing the integrated vector are then cultured in the presenceof a first selective agent that is specific for the first amplifiablemarker. This agent selects for cells that have amplified the amplifiablemarker either on the vector or on the endogenous chromosome. These cellsare then selected for amplification of the second selectable marker byculturing the cells in the presence of a second selective agent that isspecific for the second amplifiable marker. Cells that amplified thevector and flanking genomic DNA will survive this second selective step,whereas cells that amplified the endogenous first amplifiable marker orthat developed non-specific resistance will not survive. Additionalselections may be performed in similar fashion when vectors containingmore than two (e.g. three, four, five, or more) amplifiable markers areintegrated into the cell genome, by sequential culturing of the cells inthe presence of selective agents that are specific for the additionalamplifiable markers contained on the integrated vector. Followingselection, surviving cells are assayed for level of expression of adesired gene, and the cells expressing the highest levels are chosen forfurther amplification. Alternatively, pools of cells resistant to both(if two amplifiable markers are used) or all (if more than twoamplifiable markers are used) of the selective agents may be furthercultured without isolation of individual clones. These cells are thenexpanded and cultured in the presence of higher concentrations of thefirst selective agent (usually twofold higher). The process is repeateduntil the desired expression level is obtained.

Alternatively, cells containing the integrated vector may be selectedsimultaneously for both (if two are used) or all (if more than two areused) of the amplifiable markers. Simultaneous selection is accomplishedby incorporating both selection agents (if two markers are used) or allof the selection agents (if more than two markers are used) into theselection medium in which the transfected cells are cultured. Themajority of surviving cells will have amplified the integrated vector.These clones can then be screened individually to identify the cellswith the highest expression level, or they can be carried as a pool. Ahigher concentration of each selective agent (usually twofold higher) isthen applied to the cells. Surviving cells are then assayed forexpression levels. This process is repeated until the desired expressionlevels are obtained.

By either selection strategy (i.e., simultaneous or sequentialselection), the initial concentration of selective agent is determinedindependently by titrating the agent from low concentrations with nocytotoxicity to high concentrations that result in cell death in themajority of cells. In general, a concentration that gives rise todiscrete colonies (e.g., several hundred colonies per 100,000 cellsplated) is chosen as the initial concentration.

Example 5 Isolation of cDNAs Encoding Transmembrane Proteins

pRIG8R1-CD2 (FIGS. 5A-5D; SEQ ID NO:7), pRIG8R2-CD2 (FIGS. 6A-6C; SEQ IDNO:8), and pRIG8R3-CD2 (FIGS. 7A-7C; SEQ ID NO:9) vectors contain theCMV immediate early gene promoter operably linked to an exon followed byan unpaired splice donor site. The exon on the vector encodes a signalpeptide linked to the extra-cellular domain of CD2 (lacking an in framestop codon). Each vector encodes CD2 in a different reading framerelative to the splice donor site.

To create a library of activated genes, 2×10⁷ cells were irradiated with50 rads from a ¹³⁷Cs source and electroporated with 15 μg of linearizedpRIG8R1-CD2 (SEQ ID NO:7). Separately, this was repeated withpRIG8R2-CD2 (SEQ ID NO:8), and again with pRIG8R3-CD2 (SEQ ID NO:9).Following transfection, the three groups of cells were combined andplated into 150 mm dishes at 5×10⁶ transfected cells per dish to createlibrary #1. At 24 hours post transfection, library #1 was placed under500 μg/ml G418 selection for 14 days. Drug resistant clones containingthe vector integrated into the host cell genome were combined,aliquoted, and frozen for analysis. Library #2 was created as describedabove, except that 3×10⁷ cells, 3×10⁷ cells and 1×10⁷ cells weretransfected with pRIG8R1-CD2, pRIG8R2-CD2, and pRIG8R3-CD2,respectively.

To isolate cells containing activated genes encoding integral membraneproteins, 3×10⁶ cells from each library were cultured and treated asfollows:

Cells were trypsinized using 4 mls of Trypsin-EDTA.

After the cells had released, the trypsin was neutralized by addition of8 ml of alpha MEM/10% FBS.

The cells were washed once with sterile PBS and collected bycentrifugation at 800×g for 7 minutes.

The cell pellet was resuspended in 2 ml of alpha MEM/10% FBS. 1 ml wasused for sorting while the other 1 ml was replated in alpha MEM10% FBScontaining 500 μg/ml G-418, expanded and saved.

The cells used for sorting were washed once with sterile alpha MEM/10%FBS and collected by centrifigation at 800×g for 7 minutes.

The supernatant was removed and the pellet resuspended in 1 ml of alphaMEM/10% FBS. 100 μl of these cells was removed for staining with theisotype control.

200 μl of Anti-CD2 FITC (Pharmingen catalog #30054X) was added to the900 μl of cells while 20 μl of the Mouse IgG₁ isotype control(Pharmingen catalog #33814X) was added to the 100 μl of cells. The cellswere incubated, on ice, for 20 minutes.

To the tube that contained the cells stained with the Anti-Human CD2FITC, 5 ml of PBS/1% FBS were added. To the isotope control, 900 μl ofPBS/1% FBS were added. The cells were collected by centrifugation at600×g for 6 minutes.

The supernatant from the tubes was removed. The cells that had beenstained with the isotype control were resuspended in 500 μl of alphaMEM/10% FBS, and the cells that had been stained with anti-CD2-FITC wereresuspended in 1.5 ml alpha MEM/10% FBS.

Cells were sorted through five sequential sorts on a FACS Vantage FlowCytometer (Becton Dickinson Immunocytometry Systems; Mountain View,Calif.). In each sort, the indicated percentage of total cells,representing the most strongly fluorescent cells (see below) werecollected, expanded, and resorted. HT1080 cells were sorted as anegative control. The following populations were sorted and collected ineach sort:

Library #1 Library #2 Library #3 Sort #1 500,000 cells 100,000 cells40,000 cells collected (top 10%) collected (top 10%) collected (top 10%)Sort #2 300,000 cells 220,000 cells 14,000 cells collected (top 5%)collected (top 11%) collected (top 5%) Sort #3 90,000 cells collected40,000 cells collected 120,000 cells (top 5%) (top 10%) collected (top10%) Sort #4 600,000 cells (a) 6,000 cells 280,000 cells collected (top40%) collected (top 5%); collected (b) 10,000 cells (top 13%) collected(next 5%) Sort #5 (a) 260,000 cells (a) from group (a) of (Not done)collected (top 10%); sort #4, 100,000 cells (b) 530,000 cells collected(top 10%), collected (next 25%) and 350,000 cells collected (next 35%);(b) from group (b) of sort #4, 120,000 cells collected (top 10%)

Cells from each of the final sorts for each library were expanded andstored in liquid nitrogen.

Isolation of activated genes from FACS-sorted cells

Once cells had been sorted as described above, activated endogenousgenes from the sorted cells were isolated by PCR-based cloning. One ofordinary skill will appreciate, however, that any art-known method ofcloning of genes may be equivalently used to isolate activated genesfrom FACS-sorted cells.

Genes were isolated by the following protocol:

(1) Using PolyATract System 1000 mRNA isolation kit (Promega), mRNA wasisolated from 3×10⁷ CD2+ cells (sorted 5 rounds by FACS, as describedabove) from libraries #1 and #2.

(2) After mRNA isolation, the concentration of mRNA was determined bydiluting 0.5 μl of isolated mRNA into 99.5 μl water and measuring OD²⁶⁰.21 μg of mRNA were recovered from the CD2+ cells.

(3) First strand cDNA synthesis was then carried out as follows:

(a) While the PCR machine was holding at 4° C., first strand reactionmixtures were set up by sequential addition of the following components:

41 μl DEPC-treated ddH₂O

4 μl 10 mM each dNTP

8 μl 0.1 MDTT

16 μl 5×MMLV first strand buffer (Gibco-BRL)

5 μl (10 pmol/μl) of the consensus polyadenylation site primer GD.R1(SEQ ID NO:10)*

1 μl RNAsin (Promega)

3 μl (1.25 μg/μl) mRNA.

*Note: GD.R1, 5′TTTTTTTTTTTTCGTCAGCGGCCGCATCNNNNTTTATT 3′ (SEQ IDNO:10), is a “Gene Discovery” primer for first strand cDNA synthesis ofmRNA; this primer is designed to anneal to the poly-adenylation signalAATAAA and downstream poly-A region. This primer will introduce a NotIsite into the first strand.

Once samples had been made up, they were incubated as follows:

(b) 70° for 1 min.

(c) 42° hold.

2 μl of 400 U/μl SuperScript II (Gibco-BRL, Rockville, Md.) was thenadded to each sample, to give a final total volume of 82 μl. Afterapproximately three minutes, samples were incubated as follows:

(d) 37° for 30 min.

(e) 94° for 2 min.

(f) 4° for 5 min.

2 μl of 20 U/μl RNace-IT (Stratagene) was then added to each sample, andsamples were incubated at 37° for 10 min.

(4) Following first strand synthesis, cDNA was purified using a PCRcleanup kit (Qiagen) as follows:

(a) 80 μl of the first strand reaction were transferred to a 1.7 mlsiliconized eppendorf tube and adding 400 μl of PB.

(b) Samples were then transferred to a PCR clean-up column andcentrifuged for two minutes at 14,000 RPM.

(c) Columns were then disassembled, flow through decanted, 750 of μl PEwere added to pellets, and tubes were centrifuged for two minutes at14,000 RPM.

(d) Columns were disassembled and flow through decanted, and tubes thencentrifuged for two minutes at 14,000 RPM to dry resin.

(e) cDNA was then eluted using 50 μl of EB through transferring columnto a new siliconized eppendorf tube which was then centrifuged for twominutes at 14,000 RPM.

(5) Second strand cDNA synthesis was then carried out as follows:

(a) Second strand reaction mixtures were set up at RT, through thesequential addition of the following components:

ddH₂O 55 μl 10 × PCR buffer 10 μl 50 mM MgCl₂  5 μl 10 mM dNTPs  2 μl 25pmol/μl RIG.751-Bio*  4 μl 25 pmol/μl GD.R2**  4 μl First strand product20 μl *Note: RIG.F751-Bio, 5′ Biotin-CAGATCACTAGAAGCTTTATTGCGG 3′ (SEQID NO:11), anneals at the cap-site of the transcript expressed from pRIGvectors. **Note: GD.R2, 5′ TTTTCGTCAGCGGCCGCATC 3′ (SEQ ID NO:12), is aprimer used to PCR amplify cDNAs generated using primer GD.R1 (SEQ IDNO:10). GD.R2 is a sub-sequence of GD.R1 with matching sequence up tothe degenerate bases preceding the polyA signal sequence.

(b) Start second strand synthesis:

94° C for 1 min;

add 1 μl Taq (5UI/μl, Gibco-BRL);

add 1 μl Vent DNA pol (0.1 U/μl, New England Biolabs).

(c) Incubate at 63° C. for 2 min.

(d) Incubate at 72° C. for 3 min.

(e) Repeat step (b) four times.

(f) Incubate at 72° C. for 6 min.

(g) Incubate at 4° C. (hold)

(h) END

(6) 200 μl of 1 mg/ml Streptavidin-Paramagnetic Particles (SA-PMP) werethen prepared by washing three times with STE.

(7) The products of the second strand reaction were added directly tothe SA-PMPs and incubated at RT for 30 minutes.

(8) After binding, SA-PMPs were collected through the use of the magnet,and flow through material recovered.

(9) Beads were washed three times with 500 μl STE.

(10) Beads were resuspended in 50 μl of STE and collected at the bottomof the tube using the magnet. STE supernatant was then carefullypipetted off.

(11) Beads were resuspended in 50 μl of ddH₂O and placed into a 100° C.water bath for two minutes, to release purified cDNA from PMPs.

(12) Purified cDNA was recovered by collecting PMPs on the magnet andcarefully removing the supernatant containing the cDNA.

(13) Purified products were transferred to a clean tube and centrifugedat 14,000 RPM for two minutes to remove all of the residual PMPs.

(14) A PCR reaction was then carried out to specifically amplify RIGactivated cDNAs, as follows:

(a) PCR reaction mixtures were set up at RT, through the sequentialaddition of the following components:

H₂O 59 μl 10 × PCR buffer 10 μl 50 mM MgCl₂  5 μl 10 mM dNTPs  2 μl 25pmol/μl RIG.F781*  2 μl 25 pmol/μl GD.R2  2 μl second strand product 20μl *Note: RIG.F781, 5′ ACTCATAGGCCATAGAGGCCTATCACAGTTAAATTGCTAACGCAG 3′(SEQ ID NO:13), anneasl downstream of GD.F1 GD.F3, GD.F5-Bio, andRIG.F751-Bio, and adds an Sfil site for 5′ cloning of cDNAs. This primeris used in nested PCR amplification of RIG Exon1specific second strandcDNAs.

(b) Start thermal cycler:

94° C. for 3 min;

add 1 μl of Taq (5 U/μl; Gibco-BRL);

add 1 μl of 0.1 U/μl Vent DNA polymerase (New England Biolabs)

PCR was then carried out by 10 cycles of steps (c) to (e):

(c) 94° C. for 30 sec.

(d) 60° C. for 40 sec.

(e) 72° C. for 3 min.

PCR was then completed by carrying out the following steps:

(f) 94° C. for 30 sec.

(g) 60° C. for 40 sec.

(h) 72° C. for 3 min.

(i) 72° C. +20 sec each cycle for 10 cycles

(j) 72° C. for 5 min

(k) 4° C. hold.

(15) After elution of library material with 50 μl EB, samples weredigested by adding 10 μl of NEB Buffer 2, 40 μl of dH₂O and 2 μl of SfiIand digesting for 1 hour at 50° C., to cut the 5′ end of the cDNA at theSfiI site encoded by the forward primer (RIG.F781; SEQ ID NO:13).

(16) Following SfiI digestion, 5 μl of 1M NaCl and 2 μl of NotI wereadded to each sample, and samples digested for one hour at 37° C., tocut the 3′ end of the cDNA at the NotI site encoded by the first strandprimer (GD.R1;

SEQ ID NO:10).

(17) The digested cDNA was then separated on a 1% low melt agarose gel.cDNAs ranging in size from 1.2 Kb to 8 Kb were excised from the gel.

(18) cDNA was recovered from the excised agarose gel using Qiaex II GelExtraction (Qiagen). 2 μl of cDNA (approximately 30 mg) was ligated to 7μl (35 ng) of pBS-HSB (linearized with SfiI/NotI) in a total volume of10 μl of 1× T4 ligase buffer (NEB), using 400 units of T4 DNA ligase(NEB).

(19) 0.5 μl of the ligation reaction mixture from step (18) wastransformed into E. coli DH10B.

(20) 103 colonies/0.5 μl ligated DNA were recovered.

(21) These colonies were screened for exons using the primers M13F20 andJH182 (RIG Exon1 specific) through PCR in 12.5 μl volumes as follows:

(a) 100 μl of LB (with selective antibiotic) were dispensed into theappropriate number of 96-well plates.

(b) Single colonies were picked and inoculated into individual wells ofthe 96-well plate, and the plate placed into a 37° C. incubator for 2-3hours without shaking.

(c) A PCR reaction “master mix” was prepared on ice, as follows:

# of 96-Well Plates: Total # of 12.5 μl PCR 1 Plate 2 Plates 3 Plates 4Plates rxns: 96 192 288 384 dH₂O  755 μl 1.47 ml 2.20 ml 2.94 ml 5X PCRPremix-4  250 μl  500 μl  750 μl  1.0 ml F Primers premix (25   10 μl  20 μl   30 μl   40 μl pmol/μl) R Primers premix (25   10 μl   20 μl  30 μl   40 μl pmol/μl) RNace-It Cocktail  3.2 μl   63 μl  9.6 μl 12.8μl Taq Polymerase (5  3.2 μl  6.3 μl  9.6 μl 12.8 μl U/μl) Total Volume(ml) 1.01 2.02 3.03 4.04

(d) 10 μl of the master mix were dispensed into each well of the PCRreaction plate.

(e) 2.5 μl from each 100 μl E. coli culture were transferred into thecorresponding wells of the PCR reaction plate.

(f) PCR was performed, using typical PCR cycle conditions of:

(i) 94° C./2 min. (Bacterial lysis and plasmid denaturation)

(ii) 30 cycles of 92° C. denaturation for 15 sec; 60° C. primerannealing for 20 sec; and 72° C. primer extension for 40 sec

(iii) 72° C. final extension for 5 min.

(iv) 4° C. hold.

(g) Bromophenol blue was then added to the PCR reaction; samples weremixed, centrifuged, and then the entire reaction mix was loaded onto anagarose gel.

23) Of 200 clones screened, 78% were positive for the vector exon. 96 ofthese clones were grown as minipreps and purified using a Qiagen 96-wellturbo-prep following the Qiagen Miniprep Handbook (April 1997).

24) Many duplicate clones were eliminated though simultaneous digestionof 2 μl of DNA with NotI, Bam HI, XhoI, XbaI, HindIII, EcoRI in NEBBuffer 3, in a total volume of 22 μl, followed by electrophoresis on a1% agarose gel.

Results:

Two different cDNA libraries were screened using this protocol. In thefirst library (TMT#1), eight of the isolated activated genes weresequenced. Of these eight genes, four genes encoded known integralmembrane proteins and six were novel genes. In the second library(TMT#2), 11 isolated activated genes were sequenced. Of these 11 genes,one gene encoded a known integral membrane protein, one gene encoded apartially sequenced gene homologous to an integral membrane protein, andnine were novel genes. In all cases where the isolated gene correspondto a characterized known gene, that gene was an integral membraneprotein.

Exemplary significant alignments (obtained from GenBank) for genesisolated from each library are shown below:

TMT#1 Significant Alignments:

179761|gb|M76559|HUMCACNLB Human neuronal DHP-sensitive

voltage-dependent, calcium channel alpha-2b subunit mRNA

complete CDs.

Length=3600

>gi|3183974|emb|Y10183|HSMEMD H.sapiens mRNA for MEMD protein

Length=4235

TMT#2 Significant Alignments:

>gi|476590|gb|U06715|HSU06715 Human cytochrome B561, HCYTO BS61, mRNA,

partial CDs.

Length=2463

>gi|2184843|gb|AA459959|AA459959 zx66c01.s1 Soares total fetus

Nb2HF8 9w Homo sapiens cDNA clone 796414 3′ similar to

gb:J03171 INTERFERON-ALPHA RECEPTOR PRECURSOR (HUMAN);

Length=431

Example 6 Activation of Endogenous Genes using a Poly(A) Trap Vector

HT1080 cells (1×10⁷ cells) were irradiated with 50 rads using a ¹³⁷Cssource and electroporated with 15 μg linearized pRIG14 (FIGS. 29A-29B.Following transfection, the cells were plated into a 150 mm dish at5×106 cells/dish. At 24 hours, puromycin was added to 3 μg/ml. The cellswere incubated at 37° C. for 12 days in the presence of 3 μg/mlpuromycin. The media was replaced every 5 days. At 12 days, the numberof colonies was counted, and the cells were trypsinized and replatedonto a new dish. The cells were grown to 90% confluency and harvestedfor frozen storage and gene isolation. Typically, 1000-3000 colonieswere produced per 1×10⁷ cells transfected.

Example 7 Activation of Endogenous Genes Using a Dual Poly(A) Trap/SATVector

1×10⁷ HH1 cells (HPRT-minus HT1080 cells) were irradiated with 50 radsusing a ¹³⁷Cs source and electroporated with 15 μg linearized pRIG-22.Following transfection, the cells were plated into a 150 mm dish at5×10⁶ cells/dish. At 24 hours, neomycin was added to 500 μg/ml G481. Thecells were incubated at 37° C. for 4 days in the presence of 500 μg/mlG418. The media was replaced with fresh media containing 500 μg/ml G418and AgThg and grown in the presence of both drugs for an additional 7days. Alternatively, as a control for UPRT activity, the media wasreplaced with fresh media containing 500 μg/ml G418 and HAT (availablefrom Life Technologies, Inc., Rockville, Md., and used at manufacturer'srecommended concentration) and grown in the presence of both drugs foran additional 7 days. At 12 days post transfection, the number ofcolonies was counted, and the cells were trypsinized and replated onto anew dish. The cells were grown to 90% confluency and harvested forfrozen storage and gene isolation. Typically, cells subjected toG418/AgThg selection produced 1000-3000 colonies per 1×10⁷ cellstransfected. In contrast, cells subjected to G418/HAT selection producedapproximated 100 colonies per 1×10⁷ cells transfected.

Example 8 Isolation of Activated Genes

Non-targeted gene activation vectors are integrated into the genome of aeukaryotic cells using the methods of the invention. By integrating thevector into multiple cells, a library is created in which cells areexpressing different vector activated genes. RNA is isolated from thesecells using a commercial RNA isolation kit. In this example, RNA isisolated from cells using Poly(A) Tract 1000 (Promega). The RNA isconverted into cDNA, amplified, size fractionated, and cloned into aplasmid for analysis and sequencing. A brief description of this processis presented.

1) Place 4 ml GTC Extraction buffer (Poly(A) tract 1000 Kit-Promega) ina 15 ml polycarbonate screw cap tube and add 168 μl 2-mercaptoethanoland place in a 70° C. water bath.

2) Place 8 ml dilution buffer in a 15 ml polycarbonate screw cap tubefor every pellet processed and add 168 μl 2-mercaptoethanol and place ina 70° C. water bath.

3) Remove from −80° C. storage cell pellets (1×10⁷−1×10⁸ cells)containing non-targeted gene activation vector integrated into theirgenome. Pipette 4 ml GTC Extraction buffer immediately onto cell pellet.Pipette up-and-down several times until the pellet is resuspended andtransfer into a 15 ml snap cap polypropylene tube.

4) Add the 8 ml dilution buffer and mix by inversion.

5) Add 10 μl (500 pmol) of the biotinlylated oligo dT primer and mix.

6) Let sit at 70° C. for 5 minutes inverting every couple of minutes toensure even heating.

7) Centrifuge in a Sorvall HB-6 rotor at 7800 rpm (10 k×g) at 25° C. for10 minutes. During this period of time wash 6 mlStrepavidin-Paramagnetic particles (SA-PMPs) 3× with 6 ml 0.5×SSCthrough use of the Poly(A) Tract system 1000 magnet.

8) After 3 washes resuspend the SA-PMPs in 6 ml 0.5×SSC.

9) Pipette to remove the supernatant from the RNA prep and add to theresuspended SA-PMPs (Be careful when removing supernatant so that you donot disrupt the pellet).

10) Let the SA-PMP/RNA mix and incubate for 2 minutes at roomtemperature.

11) Capture the magnetic beads through use of the Poly(A) Tract system1000 magnet. Note that it takes some time for all of the beads to pelletdue to the high viscosity of the liquid.

12) Pour off the supernatant and resuspend the beads in 1.7 ml of0.5×SSC using a 2 ml pipette and transfer to a 2 ml screw cap tube.

13) Capture the SA-PMPs using the magnet and remove the supernatant bypipetting with a P1000.

14) Add 1.7 ml 0.5×SSC and invert the tube several times to mix.

15) Repeat steps 14 and 15 two more times.

16) Resuspend the SA-PMPs in 1 ml of nuclease free water and invertseveral times to mix.

17) Capture the SA-PMPs and pipette off the mRNA.

18) Place 0.5 ml of the mRNA into each of two siliconized eppendorftubes and add 50 μl of DEPC-treated 3M NaOAc solution and 0.55 ml ofisopropanol. Invert several times to mix and place at −20° C. for atleast 4 hours.

19) Centrifuge the mRNA for 10 minutes at max RPM (14 k).

20) Carefully pipette off the supernatants and wash pellets with 200 μl80% ethanol through re-centrifugation for 2 minutes at 14K RPM. Notethat the pellets are often brown or tan in color. This color resultsfrom residual SA-PMPs.

21) Remove wash and let pellets air dry for not more than 10 minutes atroom temperature.

22) Resuspend pellets in 5 μl each and combine into a single tube.

23) Centrifuge at 14K RPM for 2 minutes to remove the residual SA-PMPsand carefully remove the mRNA.

24) Determine the concentration of mRNA by diluting 0.5 μl into 99.5 μlwater and measuring OD 260. Note that 1 OD 260=40 μg RNA.

25) Set up first strand reaction for both the test sample and thenegative control (HT1080) through the sequential addition of thefollowing components while the PCR machine is holding at 4° C.:

Step 1:

42 μl DEPC-treated ddH₂O

4 μl 10 mM each dNTP

8 μl MDTT

16 μl 5×MMLV 1st strand buffer

5 μl (10 pmol/μl ) GDR1

1 μl RNAsin (Promega)

4 μl (1.25 μg/μl) mRNA.

Step 2: 70°/1 min

Step 3: 42°/hold

Step 4: After 1 minute add 2 μl SUPERSCRIPT II® (Life Technologies,Inc.; Rockville, Md.) and incubate at 37° C. for 30 min

Step 5: 94°/2 min

Step 6: 4°/∞

Step 7: Add 2 μl RNase and incubate at 37° C. for 10 min

Step 8: 4°/∞

26) Analyze 8 μl of cDNA on a 1% agarose gel to check for cDNA synthesisand purify remaining cDNA using the PCR cleanup kit from Qiagen bytransferring the 70 μl first strand reaction to a 1.5 ml siliconizedeppendorf tube and adding 400 μl PB.

27) Transfer to a PCR clean-up column and centrifuge 2 minutes at maxRPM.

28) Disassemble column and pour out Flow through. Add 750 μl PE andcentrifuge 2 minutes at max RPM.

29) Disassemble column and pour out Flow throught then centrifuge 2minutes at max RPM to dry resin.

30) Elute using 50 μl of EB through transferring column to a newsiliconized eppendorf tube and centrifuging for 2 minutes at max RPM.

31) Second Strand cDNA synthesis set up at RT:

H₂O 8.5 μl 10 × PCR buffer   5 μl 50 mM MgCl₂ 2.5 μl 10 mM dNTPs   1 μl25 pmol/μl GDF5Bio  10 μl 25 pmol/μl GDR2  10 μl First strand product 15 μl

Step 9: 94° C./1 min.

Step 10: 60° C./10 min. Add 0.25 μl Taq polymerase

Step 11: 60° C./2 min.

Step 12: 72° C./10 min.

Step 13: 94° C./1 min.

Step 14: min go to “Step 11” four more times

Step 15: 60° C./2 min

Step 16: 72° C./10 min

Step 17: END

32) Prepare 100 μl of SA-PMPs by washing 3× with STE and collectionusing a magnet. After the final wash, resuspend the beads in 150 μl STE.

33) Purify the products of the second strand reaction using the PCRcleanup kit from Qiagen. Elute in 50 μl EB and add he products of thesecond strand reaction to 150 μl of the PMPs.

34) Mix gently at RT for 30 minutes.

35) After binding collect SA-PMPs through use of a magnet and recoverflow through material (SAVE THIS MATERIAL!)

36) Wash the beads 3× with 500 μl STE and 1× with NEB 2 (1×).

37) Resuspend the beads in 100 μl NEB 2 (1×).

38) Add 2 μl SfiI and digest at 50° C. for 30 minutes with gentle mixingevery 10 minutes.

39) Recover purified cDNA through use of a magnet and carefully removingthe supernatant.

40) Transfer the products to a new tube and centrifuge at maximum RPMfor 2 minutes to remove all of the beads.

41) Set up a PCR reaction to specifically amplify RAGE activated cDNAs:

H₂O 37 μl 10 × PCR buffer 10 μl 10 mM dNTPs  2 μl 25 pmol/μl GDF 781 10μl 25 pmol/μl GDR2 10 μl Second strand product 25 μl

Step 1: 94° C./2 min.

Step 2: 94° C./45 sec.

Step 3: 60° C./10 min. Add 0.5 μl Taq Polymerase

Step 4: 72° C./10 min.

Step 6: 60° C./2 min.

Step 7: 72° C./10 min.

Step 8: Cycle to step 5, 8 more times

Step 9: 94° C./45 sec.

Step 10: 60° C./2 min.

Step 11: 72° C./10 min. +20 sec each cycle

Step12: Cycle to step 9, 14 more times

Step 13: 72° C./5 min.

Step 14: 4° C. hold

42) Check specificity of PCR amplification of HT1080 versus librarymaterial through analysis on a 1% agarose gel. If there is a highspecificity of cDNA amplification, then use Qiagen PCR clean up kit topurify PCR products.

43) After elution of library material with 50 μl EB add 10 μl NEB2, 40μl dH₂O and 2 μl SfiI and digest for 1 hour at 50° C.

44) Add 5 μl of 1 M NaCl and 2 μl of NotI and digest for 1 hour at 37°C.

45) Prepare and run a 1% L.M. agarose gel and run library material ongel. After visualization of material, cut out fragments ranging in sizefrom 500 bp to 10 Kb.

46) Recover the library DNA from agarose using Qiaex II Gel ExtractionProtocol (Qiagen) and elute DNA in 10 μl EB. Ligate 5 μl of thismaterial to 4 μl pBS-HSB (SfiI/NotI) or pBS-SNS in a total volume of 10μl.

47) Transform E. coli with 0.5 μl ligated DNA per 40 μl cells.

48) Pick colonies, grow overnight in LB, isolate plasmids.

49) Analyze gene activated cDNA inserts by restriction digest and DNAsequencing.

Example 9 Isolation of Activated Genes from Subtracted cDNA Pools

Purified mRNAs from non-transfected HT1080 cells was prepared using thePoly-A Tract 1000 system (Promega), as described in Example 8 steps1-24, and were biotinylated using EZ-Link™ Biotin LC-ASA reagent(Pierce), as follows:

1.) 25 μl DEPC-treated dH₂O and 15 μl containing 10 μg of HT1080 mRNAwas added into a siliconized microfuge tube and held on ice.

2.) Working under subdued light, 40 μl of prepared LC-ASA stock reagent(1 mg/ml in 100% ethanol) was added into the reaction tube.

3.) A UV light (365 nm wavelength) was positioned 5 cm above themicrofuge tube and used to irradiate the reaction mix for 15 minutes.

4.) Unlinked biotin reagent was removed from the labeled HT1080 mRNA bypassing the reaction mix through an RNase-free MicroSpin P-30 column(BioRad), as prescribed by the manufacturer.

HT1080 cells were transfected with a poly(A) trap pRIG activation vectorand grown under selective media to produce a population of drugresistant colonies, as described in Example 1. Purified mRNAs wereprepared from the pooled colonies using the Promega Poly-A Tract 1000system, as described in Example 8. First strand cDNA was prepared from 5μg of this mRNA using oligo GD.R1(TTTTTTTTTTTCGTCAGCGGCCGCATCNNNNTTTATT) (SEQ ID NO:10), as described inExample 8, Step 25. The reaction mix was passed through a Qiagen PCRQuick Clean-up column and the purified 1st strand cDNA was recovered in100 μl EB.

The subtractive hybridization of biotinylated HT1080 mRNAs (subtractorpopulation) and 1st strand cDNAs prepared from the superpool ofpRIG-transfected colonies (target population) was performed as follows:

1.) 9 μg of biotinylated mRNA was added into a 0.5 ml microfuge tubecontaining 0.5 μg 1st strand cDNA.

2.) 1/100× volume of 10 mg/ml glycogen, 1/10× volume of 3 M sodiumacetate, pH 5.5, and 2.6× volume of 100% ethanol were added into thetube and mixed.

3.) The tube was placed at −80° C. for 1 hr, then spun in a refrigeratedmicrofuge for 20 minutes.

4.) The pellet of precipitated nucleic acids was drained, washed oncewith 70% ethanol, then air-dried.

5.) The pellet was solvated in 5 μl HBS (50 mM HEPES, pH 7.6; 2 mM EDTA;0.2% SDS; 500 mM NaCl) and overlayered with 5 μl light mineral oil, thenheated to 95° C. for 2 minutes followed by 68° C. for 24 hours.

6.) The reaction mix was diluted with 100 μl HB (HBS without SDS) andextracted once with 100 μl chloroform to remove the oil.

7.) The diluted hybridization mix was added to 300 μlstreptavidin-coated paramagnetic particles (Promega) which had beenpre-washed 3× in 300 μl HB.

8.) The mix was incubated 10 minutes at room temperature and theSA-PMP's and bound Biotin-mRNA:DNA hybrids were removed from solution bymagnetic capture.

9.) Steps 7 and 8 were repeated once.

10.) The cleared solution was subjected to one additional round ofsubtractive hybridization and magnetic removal of captured hybrids(Steps 1-9), with the following exceptions:

Step 6: the hybridization reaction was diluted with 2×PCR Buffer (40 mMTris-HCl, pH 8.4; 100 mM KCl).

Step 7: PMPs were pre-washed in 1×PCR Buffer

The twice-subtracted 1st strand cDNA was used to generate 2nd strandcDNA by combining 45 μl of 1st strand cDNA with 7 μl dH₂O, 5 μl 50 mMMgCl₂, 2 μl premix of 10 mM each dNTP, 1 μl 10×PCR Buffer, 20 μl of 12.5pmol/μl GD19F1-Bio (5′Biotin-CTCGTTTAGTGCGGCCGCTCAGATCACTGAATTCTGACGACCT) (SEQ ID NO:14), 20μl of 12.5 pmol/μl GD.R2 (TTTTCGTCAGCGGCCGCATC) (SEQ ID NO:12), and 0.5μl Taq Polymerase, with thermocycling as described in Example 8, Step31. The second strand cDNA product was amplified and further processedfor the production of an E. coli-based cDNA library, as described inExample 8, steps 32-49.

Example 10 Selective Capture of RIG-activated Transcripts

HT1080 cells were transfected with pRIG19 activation vector (FIGS.30A-30C) and cultured for 2 weeks in selective media, as described inExample 6. Total RNA was prepared from a pellet comprised of 10⁸ cellsusing TRIzol® Reagent (Life Technologies, Inc.; Rockville, Md.)following the manufacturer's protocol, and was dissolved in 720 μl ofDEPC-treated dH₂O (dH₂O^(DEPC)). Contaminating genomic DNA waseliminated from the RNA preparation by mixing 80 μl NEB 10× Buffer 2, 8μl Promega RNasin, and 20 μl RQ1 Promega RNase-free DNase, incubating at37° C. for 30 minutes, extracting sequentially with equal volumes ofphenol:chlorofom(1:1) and chloroform, mixing with 1/10× volume sodiumacetate (pH 5.5), precipitating the RNA with 2× volume of 100% ethanol,and solvating the dried RNA pellet in dH₂O^(DEPC) to a finalconcentration of 4.8 μg/μl.

mRNA transcripts derived from pRIG19-activated genes were selectivelycaptured from the pool of total cellular RNAs by mixing in a 2 mlRNase-free microfuge tube 150 μl total RNA, 150 μl HBDEPC (50 mM HEPES,pH 7.6; 2 mM EDTA; 500 mM NaCl), 3 μl Promega RNasin, and 2.5 μl (25pmol/μl) oligo GD19.R1-Bio (see Table 1), then incubating at 70° C. for5 minutes followed by 50° C. for 15 minutes. One ml of Promegastreptavidin coated paramagnetic particles (SA-PMPs) was magneticallycaptured and washed 3× each with 1.5 ml of 0.5×SSC, and the SA-PMPs wereleft without being resuspended. The warm oligo:RNA hybridizationreaction was added directly into the tube containing the semi-drySA-PMPs. After incubating for 10 minutes at room temperature the SA-PMPswere washed 3× with 1 ml 0.5×SSC.

TABLE 1 Primer and Oligonucleotide Sequences SEQ Primer/Oligo ID NameSequence NO: Forward GD19.F1-Bio 5′Biotin-CTCGTTTAGTGCGG- 14 PCRCCGCTCAGATCACTGAATTC Primers TGACGACCT GDl9.F2-Bio5′Biotin-CTCGTTTAGTGGCG- 15 CGCCAGATCACTGAATTCTG ACGACCT GDl9.F2GACCTACTGATTAACGGCC- 16 ATA Reverse GD.R1 TTTTTTTTTTTTCGTCAGCG- 10 PCRGCCGCATCNNNNTTTATT Primers GD.R2 TTTTCGTCAGCGGCCGCATC 12 mRNAGD19.R1-Bio TCGTCAGAATTCAGTGAT- 17 Capture CT-3′ Biotin Oligo

After the final magnetic capture, the SA-PMP's were suspended in 190 μldH₂ODEPC and incubated at 68° C. for 15 minutes. PMPs were immobilizedby exposure to a magnetic and the cleared solution containingRIG-activated transcripts was transferred to a microfuge tube. 63 μl ofcaptured RIG-activated transcript were transferred to a PCR tube wherefirst and second strand cDNA synthesis was performed using PCR program“1+2CDNA”, as follows:

Step1: 4° C./∞: Add into the PCR tube containing the RIG-activatedtranscripts 20 μl 5× GibcoBRL RT Buffer, 1 μl Promega RNasin, 10 μl 100mM DTT, 5 μl dNTP premix at 10 mM each, 1 μl oligo GD.R1 (see Table 1)at 25 pmol/μl.

Step 2: 70° C./3 minutes

Step 3: 42° C./10 minutes

Step 4. Add 2.5 μl SUPERSCRIPT II® (Life Technologies, Inc.), thenincubate at 37° C./1 hour

Step 5: 94° C./2 minutes

Step 6: 4° C./∞.

To the 1st strand cDNA mix, 2 μl of Stratagene RNase-It was added andthe mixture was incubated at 37° C. for 15 minutes. 600 μl of Qiagen PBreagent was added to the reaction, then transferred to a Qiagen PCRclean-up column and processed according to the manufacturer's protocol.cDNA was eluted from the column in 50 μl EB and transferred to a PCRtube. The second strand cDNA reaction was performed using oligosGD19.F2-Bio (Table 1) and GD.R2 (Table 1) as described in Example 9. Thesecond strand product was captured on Promega SA-PMPs as described inExample 9, with the exception that the final suspension of SA-PMPs wasin 1× NEB 4 Buffer and the captured cDNAs were cleaved from theparticles using restriction endonuclease Asc I. Amplification of thesecond strand cDNA products using oligos GD19.F2 and GD.R2, digestion ofthe amplified cDNAs using endonucleases SfiI and NotI, and sizeselection of cDNAs prior to cloning were all performed as described inExample 9. The final cDNA cleanup was achieved by eluting the cDNA pooloff a Qiagen PCR Cleanup column in 30 μl EB. 11 μl of cDNA was mixedwith 4 μl 5× GibcoBRL Ligase Buffer, 4 μl pGD5 vector DNA previouslyprepared by digestion with SfiI, NotI, and CIP. 1 μl T4 DNA Ligase wasadded, and the reaction nix was incubated at 16° C. overnight. 1 μl ofligation reaction was used to transform electro-competent E. coli DH10Bcells, which were subsequently plated on LB agar plates containing 12.5μg/ml chloramphenicol. Typically, 60 to 80 bacterial colonies wererecovered per μl of ligation mix transformed.

Example 11 Selective Capture of RIG-activated Transcripts

HT1080 cells were transfected with pRIG19 activation vector and culturedfor 2 weeks in selective media, as described in Example 6. Total RNA wasprepared from a pellet comprised of 10⁸ cells using TRIzol® Reagent(Life Technologies, Inc.) following the manufacturer's protocol, and wasdissolved in 720 μl of DEPC treated dH₂O (dH₂O^(DEPC)). Contaminatinggenomic DNA was eliminated from the RNA preparation by mixing 80 μl NEB10× Buffer 2, 8 μl Promega RNasin, and 20 μl RQ1 Promega RNase-freeDNase, incubating at 37° C. for 30 minutes, extracting sequentially withequal volumes of phenol:chlorofom (1:1) and chloroform, mixing with1/10× volume sodium acetate (pH 5.5), precipitating the RNA with 2×volume of 100% ethanol, and solvating the dried RNA pellet in dH2ODEPCto a final concentration of 4.8 μg/μl.

mRNA transcripts derived from pRIG19-activated genes were selectivelycaptured from the pool of total cellular RNAs by mixing in a 2 mlRNase-free microfuge tube 150 μl total RNA, 150 μl HBDEPC (50 mM HEPES,pH 7.6; 2 mM EDTA; 500 mM NaCl), 3 μl Promega RNasin, and 2.5 μl (25pmol/μl) oligo GD19.R1-Bio (see Table 1), then incubating at 70° C. for5 minutes followed by 50° C. for 15 minutes. One ml of Promegastreptavidin coated paramagnetic particles (SA-PMPs) was magneticallycaptured and washed 3× each with 1.5 ml of 0.5×SSC, and the SA-PMPs wereleft without being resuspended. The warm oligo:RNA hybridizationreaction was added directly into the tube containing the semi-drySA-PMPs. After incubating for 10 minutes at room temperature the SA-PMPswere washed 3× with 1 ml 0.5×SSC. After the final magnetic capture theSA-PMP's were suspended in 190 μl dH₂O^(DEPC) and incubated at 68° C.for 15 minutes. PMPs were immobilized by exposure to a magnetic and thecleared solution containing RIG-activated transcripts was transferred toa microfuge tube. 63 μl of captured RIG-activated transcript weretransferred to a PCR tube where first and second strand cDNA synthesiswas performed using PCR program “1+2CDNA”, as follows:

Step1: 4° C./∞: Add into the PCR tube containing the RIG-activatedtranscripts 20 μl 5× GibcoBRL RT Buffer, 1 μl Promega RNasin, 10 μl 100mM DTT, 5 μl dNTP premix at 10 mM each, 1 μl oligo GD.R1 (see Table 1)at 25 pmol/μl.

Step 2: 70° C./3 minutes

Step 3: 42° C./10 minutes

Step 4: Add 2.5 μl SUPERSCRIPT II® (Life Technologies, Inc.), thenincubate at 37° C./1 hour

Step 5: 94° C./2 minutes

Step 6: 60° C./∞; while holding temperature, the following were added: 2μl 50 mM MgCl₂, 1 μl oligo GD19.F1-Bio (Table 1) at 25 pmol/μl, and 2 μlStratagene RNace-It. After 10 minutes, 0.5 μl Taq DNA Polymerase (LifeTechnologies, Inc.) was added and the cycling was continued:

Step 7: 72° C./10 minutes

Step 8: 4° C./∞.

The 100 μl volume cDNA reaction mix was transferred to a 1.5 mlsiliconized microfuge tube and extracted sequentially with equal volumesof phenol:chloroform (1:1) and chloroform, and the aqueous phase wastransferred to a new tube and place in speed-vac for 5 minutes at 37° C.Restriction digestion of the cDNA was performed by adding 74 μl dH₂O, 20μl NEB 10× Buffer 2, 2 μl 1 mg/ml BSA, 4 μl SfiI and incubating at 50°C. for 1 hour, then adding 10 μl 1 M NaCl, 4 μl NotI and incubating anadditional 37° C. for 1 hour. The reaction mix was extractedsequentially with equal volumes of phenol: chloroform (1:1) andchloroform, then cDNAs were precipitated by adding 1/100× volume 10mg/ml glycogen, 1/30× volume 3 M sodium acetate(pH 7.5), 2× volume 100%absolute ethanol, and freezing at −80° C. for 1 hour. The cDNA pelletwas washed once with 70% ethanol and air dried for 15 minutes, thensolvated in 5 μl dH₂O, 1 μl 10× NEB Ligase Buffer, 4 μl pGD5 vector DNApreviously prepared by digestion with SfiI, NotI, and CIP. 0.5 μl T4 DNALigase was added, and the reaction mix was incubated at 16° C.overnight. 10 μl dH₂O was added to the ligation reaction and 0.5 μl wasused to transform electro-competent E. coli DH10B cells. Typically, 6 to10 colonies per μl of transformed ligation mix were observed.

Example 12 Ligation of Activation Vectors to Genomic DNA andTransfection into Human Cells

Genomic DNA was harvested from a human cell line, HT1080 (10⁸ cells),according to published procedures (Sambrook et al., Molecular Cloning,Cold Spring Harbor Laboratory Press, (1989)). The isolated genomic DNAwas digested with BamHI under conditions that resulted in incompletedigestion. This was accomplished by titrating the amount of BamHI in thereaction. Each reaction contained 10 μg genomic DNA and BamHI at aconcentration of either 0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28,2.56, 5.62, or 11.24 units. After a one hour incubation at 37° C., thereactions were stopped by phenol extraction, followed by ethanolprecipition. The digested DNA from each reaction was separated byagarose gel electrophoresis. Reactions containing DNA predominantly inthe range of 10 kb to 400 kb were combined for ligation to theactivation vector. The pooled, digested genomic DNA was then added toBamHI linearized activation vector in 1× ligation buffer. Ligase (LifeTechnologies, Inc., 40 units) was added and the ligation reaction wasincubated at 16° C. for 24 hours. Following ligation, the genomicDNA/activation vector was transfected into HT1080 cells usingLIPOFECTIN® (Life Technologies, Inc.) according to the manufacturer'sprocedures. Optionally, the HT1080 cells were irradiated prior to orafter transfection. When cells were irradiated, doses in the range of0.1 rads to 200 rads were found to be particularly useful. Followingtransfection, cells were grown incomplete media. At 36 hourspost-transfection, G418 (300 μg/ml) were added to the media. At 10-14days post selection, the drug resistant clones were pooled, expanded,and harvested. Total RNA or mRNA was collected from the harvested cells.cDNA derived from vector activated genes was then synthesized andisolated using the methods described herein (see, e.g., Example 8supra).

Example 13 Co-transfections of BAC Contig Clones with the ActivationVector

Genomic libraries were created in pUniBAC (FIGS. 34A-34B) according topublished procedures (Shizuya et al., Proc. Natl. Acad. Sci. USA 89:8794(1992)). Typically, the size of genomic fragments can be between 1 kband 500 kb, and preferably between 50 kb and 500 kb. The BAC library waspropagated in E. coli. To prepare plasmids for transfection, the librarywas plated onto LB agar plates containing 12.5 μg/ml chloramphenicol.Approximately 1000 clones were present on each 150 mm plate. Followinggrowth and selection, the colonies from each plate were eluted from theagar plate through the addition of LB and pooled. Bach pool (˜10,000clones) was grown in 1 liter LB/12.5 μg/ml chloramphenicol overnight.BAC plasmids were then isolated from each pool using a commercial kit(Qiagen).

Purified BAC clones were digested with I-Ppo-I which cleaves a uniquesite in the BAC vector flanking the cloning site. Since I-Ppo-I is anultra-rare cutter, it will not digest the vast majority of genomic DNAinserts. Following digestion, the linearized genomic library clones werecotransfected into HT1080 cells using LIPOFECTIN® (Life Technologies,Inc.) according to the manufacturer's directions. Briefly, 10 μg of BACgenomic DNA was combined with 1 μg of linearized pRIG20 (FIGS. 31A-31C)in α-MEM (no serum). 5 μg of LIPOFECTIN® was added to the DNA and themixture was incubated at room temperature for 15 minutes. TheDNA/LIPOFECTIN® mixture was then added to 10⁵ HT1080 cells in a 6 welldish. The cells were incubated with the DNA/LIPOFECTIN® in serum freeα-MEM for 12 hours, washed, and placed in α-MEM/10% FBS for 36 hours. Toselect for cells that had integrated the vector and genomic DNA, thetransfected cells were replated into a 10 cm dish and incubated in thepresence of 300 μg/ml G418 for 10 days. Drug resistant clones wereexpanded and harvested to allow isolation of the activated cDNAmolecules as described herein in Example 8.

Example 14 In vitro Integration of Activation Vector into PurifiedGenomic DNA and Transfection of the Integration Products into Host Cells

Genomic DNA was isolated and cloned into the Bacterial ArtificialChromosome, pUniBAC (FIGS. 34A-34B), using published procedures(Sambrook et al., Molecular Cloning, Cold Spring Harbor LaboratoryPress, (1989); Shizuya et al., Proc. Natl. Acad. Sci. USA 89:8794(1992)). Following ligation of the genomic inserts into pUniBAC, theplasmids were transformed into the E. coli strain DH10B (LifeTechnologies, Inc.) and selected on tetracycline. Individual bacterialclones were combined into pools containing approximately 1000 members.Each pool was grown to saturation in 1 liter LB/tetracycline. pUniBACplasmids containing genomic DNA inserts were isolated from the bacteriausing a commercial kit (Qiagen).

For each pool of UniBAC clones, 2 μg of the library were incubated with50 ng of the activation vector pRIG-T and 1 unit of mutant Tn5transposase for 2 hours at 37° C. (transposase available from EpicentreTechnologies). Following incubation, the pUniBAC clones were transformedinto DH10B cells and selected on chloramphenicol. All colonies from eachpool were combined and grown in 1 liter LB/chloramphenicol. Plasmidswere harvested using Qiagen Tip-500 columns according to themanufacturer's instructions.

For each pool, 20 μg of the library was transfected into 2×10⁶ HT1080cells with 30 μg Ex-gen 500 (MBI Fermentas) according to themanufacturer's instructions. At 48 hours post-transfection, the cellswere placed into media containing 3 μg/ml puromycin. After 10 days ofgrowth in the presence of puromycin, drug resistant clones were pooled,expanded and harvested for gene discovery. To isolate vector activatedgenes, mRNA from each pool of cells was isolated, converted to cDNA, andcloned into plasmids as described in Example 8. Individual cDNA cloneswere analyzed by restriction digestion and sequencing.

Example 15 Creation of Protein Expression Libraries from Cloned GenomicDNA

A genomic library containing genomic DNA inserts (100 kb avg. size) wascreated in pUniBAC as described in Examples 13 and 14. (Note: In someembodiments of the invention, the genomic fragments are cloned into thelinearization site of an activation vector, wherein the activationvector is preferably a YAC, BAC, PAC, or Cosmid based vector.) In thisexample, the activation vector, pRIG-TP, was integrated into the BACgenomic library using in vitro transposition as described in Example 14.pRIG-TP is shown in FIG. 36. Following integration, the library plasmidswere transformed into E. coli and BAC vectors containing an integratedpRIG-TP vector were selected for on chloramphenicol plates. Colonieswere pooled and grown to saturation in LB/Tetracycline. BAC plasmidswere harvested using a commercial kit (Qiagen).

For each transfection, 20 ug of the BAC library was transfected into2×10⁶ HT1080 cells using 30 ug Ex-gen 500 (MBI Fermentas) according tothe manufacturer's instructions. At 48 hours post transfection, thecells were placed into mdia containing 3 ug/ml puromycin. After 10 daysof selection, drug resistant clones were pooled and expanded. Theexpaned pools of drug resistant clones were divided into separate groupsfor freezing, protein production, and episome amplification.

To isolate and test activated secreted proteins, culture supernatantswere harvested and saved at −80° C. until used in specific assays.Activated intracellular proteins were harvested from cell lysates(prepared by any method known in the art) and used in in vitro assays.

To amplify the copy number of the BAC episomes, the cells were selectedwith increasing concentrations of methotrexate. In these experiments,the initial methotrexate concentration was 20 mM. Methotrexateconcentrations were doubled every 7 days until cells resistant to 5 μMwere obtained. At each methotrexate concentration, a portion of cellswere removed for storage and protein production. Activated secreted andintracellular proteins were harvested from these cells as described forthe non-methotrexate selected cells.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

33 1 39 DNA Homo sapiens 1 tccttcgaag cttgtcatgg ttggttcgct aaactgcat 392 40 DNA Homo sapiens 2 aaacttaaga tcgattaatc attcttctca tatacttcaa 40 328 DNA Homo sapiens 3 atccaccatg gctacaggtg agtactcg 28 4 36 DNA Homosapiens 4 gatccgagta ctcacctgta gccatggtgg atttaa 36 5 33 DNA Homosapiens 5 ggcgagatct agcgctatat gcgttgatgc aat 33 6 51 DNA Homo sapiens6 ggccagatct gctaccttaa gagagccgaa acaagcgctc atgagcccga a 51 7 6084 DNAHomo sapiens 7 agatcttcaa tattggccat tagccatatt attcattggt tatatagcataaatcaatat 60 tggctattgg ccattgcata cgttgtatct atatcataat atgtacatttatattggctc 120 atgtccaata tgaccgccat gttggcattg attattgact agttattaatagtaatcaat 180 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataacttacggtaaa 240 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataatgacgtatgt 300 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagtatttacggta 360 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgccccctattgacgt 420 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttacgggactttcc 480 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgcggttttggca 540 gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtctccaccccat 600 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaaaatgtcgtaa 660 caactgcgat cgcccgcccc gttgacgcaa atgggcggta ggcgtgtacggtgggaggtc 720 tatataagca gagctcgttt agtgaaccgt cagatcacta gaagctttattgcggtagtt 780 tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctcgaacttaagc 840 tgcagtgact ctcttaatta actccaccag tctcacttca gttccttttgcctccaccag 900 tctcacttca gttccttttg catgaagagc tcagaatcaa aagaggaaaccaacccctaa 960 gatgagcttt ccatgtaaat ttgtagccag cttccttctg attttcaatgtttcttccaa 1020 aggtgcagtc tccaaagaga ttacgaatgc cttggaaacc tggggtgccttgggtcagga 1080 catcaacttg gacattccta gttttcaaat gagtgatgat attgacgatataaaatggga 1140 aaaaacttca gacaagaaaa agattgcaca attcagaaaa gagaaagagactttcaagga 1200 aaaagataca tataagctat ttaaaaatgg aactctgaaa attaagcatctgaagaccga 1260 tgatcaggat atctacaagg tatcaatata tgatacaaaa ggaaaaaatgtgttggaaaa 1320 aatatttgat ttgaagattc aagagagggt ctcaaaacca aagatctcctggacttgtat 1380 caacacaacc ctgacctgtg aggtaatgaa tggaactgac cccgaattaaacctgtatca 1440 agatgggaaa catctaaaac tttctcagag ggtcatcaca cacaagtggaccaccagcct 1500 gagtgcaaaa ttcaagtgca cagcagggaa caaagtcagc aaggaatccagtgtcgagcc 1560 tgtcagctgt ccagagaaag ggatccaggt gagtagggcc cgatccttctagagtcgagc 1620 tctcttaagg tagcaaggtt acaagacagg tttaaggaga ccaatagaaactgggcttgt 1680 cgagacagag aagactcttg cgtttctgat aggcacctat tggtcttacgcggccgcgaa 1740 ttccaagctt gagtattcta tcgtgtcacc taaataactt ggcgtaatcatggtcatatc 1800 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacgagccggaagca 1860 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaattgcgttgcgcg 1920 atgcttccat tttgtgaggg ttaatgcttc gagaagacat gataagatacattgatgagt 1980 ttggacaaac cacaacaaga atgcagtgaa aaaaatgctt tatttgtgaaatttgtgatg 2040 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaacaacaattgca 2100 ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagcaagtaaaacc 2160 tctacaaatg tggtaaaatc cgataaggat cgattccgga gcctgaatggcgaatggacg 2220 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcacgtgaccgctac 2280 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttctcgccacgtt 2340 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttccgatttagtgc 2400 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgtagtgggccatc 2460 gccctgatag acggtttttc gccctttgac gttggagtcc acgttctttaatagtggact 2520 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttgatttataagg 2580 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaaaatttaacgc 2640 gaattttaac aaaatattaa cgcttacaat ttcgcctgtg taccttctgaggcggaaaga 2700 accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctccccagcaggca 2760 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaagtccccaggct 2820 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaaccatagtcccgc 2880 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattctccgccccatg 2940 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctctgagctattcc 3000 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcttgattcttctg 3060 acacaacagt ctcgaactta aggctagagc caccatgatt gaacaagatggattgcacgc 3120 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcacaacagacaat 3180 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccggttctttttgt 3240 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgcggctatcgtg 3300 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactgaagcgggaag 3360 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctcaccttgctcc 3420 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgcttgatccggc 3480 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgtactcggatgga 3540 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcgcgccagccga 3600 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcgtgacccatgg 3660 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggattcatcgactg 3720 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctacccgtgatattgc 3780 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggtatcgccgctcc 3840 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgagcgggactctg 3900 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgatggccgcaataaaat 3960 atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaagatccgcgtatggtgca 4020 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacacccgccaacac 4080 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacagacaagctgtga 4140 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaacgcgcgagac 4200 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataataatggtttctt 4260 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgtttatttttct 4320 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatgcttcaataat 4380 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttattcccttttttg 4440 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagtaaaagatgctg 4500 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagcggtaagatcc 4560 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaagttctgctat 4620 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgccgcatacact 4680 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatcttacggatggca 4740 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacactgcggccaact 4800 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcacaacatggggg 4860 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccataccaaacgacg 4920 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaactattaactggcg 4980 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcggataaagttg 5040 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgataaatctggag 5100 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggtaagccctccc 5160 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacgaaatagacaga 5220 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaagtttactcat 5280 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctaggtgaagatcc 5340 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccactgagcgtcag 5400 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgcgtaatctgct 5460 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggatcaagagctac 5520 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaatactgtccttc 5580 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcctacatacctcg 5640 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgtcttaccgggt 5700 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacggggggttcgt 5760 gcacacagcc cagcttggag cgaacgacct acaccgaact gagatacctacagcgtgagc 5820 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccggtaagcggca 5880 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctggtatctttata 5940 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgctcgtcagggg 6000 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctggccttttgct 6060 ggccttttgc tcacatggct cgac 6084 8 6085 DNA Homo sapiens8 agatcttcaa tattggccat tagccatatt attcattggt tatatagcat aaatcaatat 60tggctattgg ccattgcata cgttgtatct atatcataat atgtacattt atattggctc 120atgtccaata tgaccgccat gttggcattg attattgact agttattaat agtaatcaat 180tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 240tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 300tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 360aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt 420caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc 480tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 540gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 600tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 660caactgcgat cgcccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 720tatataagca gagctcgttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt 780tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc 840tgcagtgact ctcttaatta actccaccag tctcacttca gttccttttg cctccaccag 900tctcacttca gttccttttg catgaagagc tcagaatcaa aagaggaaac caacccctaa 960gatgagcttt ccatgtaaat ttgtagccag cttccttctg attttcaatg tttcttccaa 1020aggtgcagtc tccaaagaga ttacgaatgc cttggaaacc tggggtgcct tgggtcagga 1080catcaacttg gacattccta gttttcaaat gagtgatgat attgacgata taaaatggga 1140aaaaacttca gacaagaaaa agattgcaca attcagaaaa gagaaagaga ctttcaagga 1200aaaagataca tataagctat ttaaaaatgg aactctgaaa attaagcatc tgaagaccga 1260tgatcaggat atctacaagg tatcaatata tgatacaaaa ggaaaaaatg tgttggaaaa 1320aatatttgat ttgaagattc aagagagggt ctcaaaacca aagatctcct ggacttgtat 1380caacacaacc ctgacctgtg aggtaatgaa tggaactgac cccgaattaa acctgtatca 1440agatgggaaa catctaaaac tttctcagag ggtcatcaca cacaagtgga ccaccagcct 1500gagtgcaaaa ttcaagtgca cagcagggaa caaagtcagc aaggaatcca gtgtcgagcc 1560tgtcagctgt ccagagaaag ggatcccagg tgagtagggc ccgatccttc tagagtcgag 1620ctctcttaag gtagcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg 1680tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac gcggccgcga 1740attccaagct tgagtattct atcgtgtcac ctaaataact tggcgtaatc atggtcatat 1800ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 1860ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 1920gatgcttcca ttttgtgagg gttaatgctt cgagaagaca tgataagata cattgatgag 1980tttggacaaa ccacaacaag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat 2040gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc 2100attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac 2160ctctacaaat gtggtaaaat ccgataagga tcgattccgg agcctgaatg gcgaatggac 2220gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcac gtgaccgcta 2280cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 2340tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 2400ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat 2460cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 2520tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag 2580ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 2640cgaattttaa caaaatatta acgcttacaa tttcgcctgt gtaccttctg aggcggaaag 2700aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc 2760agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc 2820tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg 2880cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 2940ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc 3000cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct tgattcttct 3060gacacaacag tctcgaactt aaggctagag ccaccatgat tgaacaagat ggattgcacg 3120caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 3180tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 3240tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt 3300ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 3360gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 3420ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 3480ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 3540aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 3600aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg 3660gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 3720gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 3780ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 3840ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct 3900ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgatggc cgcaataaaa 3960tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaagatccg cgtatggtgc 4020actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca 4080cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 4140accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 4200cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 4260tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 4320taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 4380tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 4440gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 4500gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 4560cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 4620tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 4680tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 4740atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 4800ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 4860gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 4920gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 4980gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 5040gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 5100gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 5160cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 5220atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 5280tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 5340ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 5400gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 5460tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 5520ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 5580ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 5640gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 5700ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 5760tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 5820ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 5880agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 5940agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 6000gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 6060tggccttttg ctcacatggc tcgac 6085 9 6086 DNA Homo sapiens 9 agatcttcaatattggccat tagccatatt attcattggt tatatagcat aaatcaatat 60 tggctattggccattgcata cgttgtatct atatcataat atgtacattt atattggctc 120 atgtccaatatgaccgccat gttggcattg attattgact agttattaat agtaatcaat 180 tacggggtcattagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 240 tggcccgcctggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 300 tcccatagtaacgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 360 aactgcccacttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt 420 caatgacggtaaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc 480 tacttggcagtacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 540 gtacaccaatgggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 600 tgacgtcaatgggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 660 caactgcgatcgcccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 720 tatataagcagagctcgttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt 780 tatcacagttaaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc 840 tgcagtgactctcttaatta actccaccag tctcacttca gttccttttg cctccaccag 900 tctcacttcagttccttttg catgaagagc tcagaatcaa aagaggaaac caacccctaa 960 gatgagctttccatgtaaat ttgtagccag cttccttctg attttcaatg tttcttccaa 1020 aggtgcagtctccaaagaga ttacgaatgc cttggaaacc tggggtgcct tgggtcagga 1080 catcaacttggacattccta gttttcaaat gagtgatgat attgacgata taaaatggga 1140 aaaaacttcagacaagaaaa agattgcaca attcagaaaa gagaaagaga ctttcaagga 1200 aaaagatacatataagctat ttaaaaatgg aactctgaaa attaagcatc tgaagaccga 1260 tgatcaggatatctacaagg tatcaatata tgatacaaaa ggaaaaaatg tgttggaaaa 1320 aatatttgatttgaagattc aagagagggt ctcaaaacca aagatctcct ggacttgtat 1380 caacacaaccctgacctgtg aggtaatgaa tggaactgac cccgaattaa acctgtatca 1440 agatgggaaacatctaaaac tttctcagag ggtcatcaca cacaagtgga ccaccagcct 1500 gagtgcaaaattcaagtgca cagcagggaa caaagtcagc aaggaatcca gtgtcgagcc 1560 tgtcagctgtccagagaaag ggatccacag gtgagtaggg cccgatcctt ctagagtcga 1620 gctctcttaaggtagcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt 1680 gtcgagacagagaagactct tgcgtttctg ataggcacct attggtctta cgcggccgcg 1740 aattccaagcttgagtattc tatcgtgtca cctaaataac ttggcgtaat catggtcata 1800 tctgtttcctgtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 1860 cataaagtgtaaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 1920 cgatgcttccattttgtgag ggttaatgct tcgagaagac atgataagat acattgatga 1980 gtttggacaaaccacaacaa gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga 2040 tgctattgctttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg 2100 cattcattttatgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa 2160 cctctacaaatgtggtaaaa tccgataagg atcgattccg gagcctgaat ggcgaatgga 2220 cgcgccctgtagcggcgcat taagcgcggc gggtgtggtg gttacgcgca cgtgaccgct 2280 acacttgccagcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 2340 ttcgccggctttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 2400 gctttacggcacctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 2460 tcgccctgatagacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 2520 ctcttgttccaaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa 2580 gggattttgccgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 2640 gcgaattttaacaaaatatt aacgcttaca atttcgcctg tgtaccttct gaggcggaaa 2700 gaaccagctgtggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg 2760 cagaagtatgcaaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg 2820 ctccccagcaggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc 2880 gcccctaactccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca 2940 tggctgactaatttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt 3000 ccagaagtagtgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttgattcttc 3060 tgacacaacagtctcgaact taaggctaga gccaccatga ttgaacaaga tggattgcac 3120 gcaggttctccggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca 3180 atcggctgctctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt 3240 gtcaagaccgacctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg 3300 tggctggccacgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga 3360 agggactggctgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct 3420 cctgccgagaaagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg 3480 gctacctgcccattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg 3540 gaagccggtcttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc 3600 gaactgttcgccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat 3660 ggcgatgcctgcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac 3720 tgtggccggctgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt 3780 gctgaagagcttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct 3840 cccgattcgcagcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc 3900 tggggttcgaaatgaccgac caagcgacgc ccaacctgcc atcacgatgg ccgcaataaa 3960 atatctttattttcattaca tctgtgtgtt ggttttttgt gtgaagatcc gcgtatggtg 4020 cactctcagtacaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac 4080 acccgctgacgcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt 4140 gaccgtctccgggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag 4200 acgaaagggcctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc 4260 ttagacgtcaggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 4320 ctaaatacattcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 4380 atattgaaaaaggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 4440 tgcggcattttgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 4500 tgaagatcagttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 4560 ccttgagagttttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 4620 atgtggcgcggtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 4680 ctattctcagaatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 4740 catgacagtaagagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 4800 cttacttctgacaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 4860 ggatcatgtaactcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 4920 cgagcgtgacaccacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 4980 cgaactacttactctagctt cccggcaaca attaatagac tggatggagg cggataaagt 5040 tgcaggaccacttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 5100 agccggtgagcgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 5160 ccgtatcgtagttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 5220 gatcgctgagataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 5280 atatatactttagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 5340 cctttttgataatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 5400 agaccccgtagaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 5460 ctgcttgcaaacaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 5520 accaactctttttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 5580 tctagtgtagccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 5640 cgctctgctaatcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 5700 gttggactcaagacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 5760 gtgcacacagcccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 5820 gctatgagaaagcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 5880 cagggtcggaacaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 5940 tagtcctgtcgggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 6000 ggggcggagcctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 6060 ctggccttttgctcacatgg ctcgac 6086 10 38 DNA Artificial Sequence modified_base(29)..(32) a, c, t, g, other or unknown 10 tttttttttt ttcgtcagcggccgcatcnn nntttatt 38 11 25 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 11 cagatcacta gaagctttattgcgg 25 12 20 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 12 ttttcgtcag cggccgcatc 20 13 45 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 13 actcataggc catagaggcc tatcacagtt aaattgctaa cgcag 4514 43 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 14 ctcgtttagt gcggccgctc agatcactga attctgacgacct 43 15 41 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 15 ctcgtttagt ggcgcgccag atcactgaat tctgacgacct 41 16 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 16 gacctactga ttaacggcca ta 22 17 20 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 17 tcgtcagaat tcagtgatct 20 18 6836 DNA Homo sapiens 18agatcttcaa tattggccat tagccatatt attcattggt tatatagcat aaatcaatat 60tggctattgg ccattgcata cgttgtatct atatcataat atgtacattt atattggctc 120atgtccaata tgaccgccat gttggcattg attattgact agttattaat agtaatcaat 180tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 240tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 300tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 360aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt 420caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc 480tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 540gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 600tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 660caactgcgat cgcccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 720tatataagca gagctcgttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt 780tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc 840tgcagtgact ctcttaaatc caccatggct acaggtgagt actcggatct agcgctatat 900gcgttgatgc aatttctatg cgcacccgtt ctcggagcac tgtccgaccg ctttggccgc 960cgcccagtcc tgctcgcttc gctacttgga gccactatcg actacgcgat catggcgacc 1020acacccgtcc tgtggatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca 1080ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac 1140ttcgggctca tgagcgcttg tttcggctct cttaaggtag cagatccttg ctagagtcga 1200ccaattctca tgtttgacag cttatcatcg cagatcctga gcttgtatgg tgcactctca 1260gtacaatctg ctctgctgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg 1320aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca 1380attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca 1440gatatacgcg tatctgaggg gactagggtg tgtttaggcg cccagcgggg cttcggttgt 1500acgcggttag gagtcccctc aggatatagt agtttcgctt ttgcataggg agggggaaat 1560gtagtcttat gcaatacact tgtagtcttg caacatggta acgatgagtt agcaacatgc 1620cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg 1680tgccttatta ggaaggcaac agacaggtct gacatggatt ggacgaacca ctgaattccg 1740cattgcagag ataattgtat ttaagtgcct agctcgatac aataaacgcc atttgaccat 1800tcaccacatt ggtgtgcacc tccaagctgg gtaccagctg ctagcctcga gacgcgtgat 1860ttccttcgaa gcttgtcatg gttggttcgc taaactgcat cgtcgctgtg tcccagaaca 1920tgggcatcgg caagaacggg gacctgccct ggccaccgct caggaatgaa ttcagatatt 1980tccagagaat gaccacaacc tcttcagtag aaggtaaaca gaatctggtg attatgggta 2040agaagacctg gttctccatt cctgagaaga atcgaccttt aaagggtaga attaatttag 2100ttctcagcag agaactcaag gaacctccac aaggagctca ttttctttcc agaagtctag 2160atgatgcctt aaaacttact gaacaaccag aattagcaaa taaagtagac atggtctgga 2220tagttggtgg cagttctgtt tataaggaag ccatgaatca cccaggccat cttaaactat 2280ttgtgacaag gatcatgcaa gactttgaaa gtgacacgtt ttttccagaa attgatttgg 2340agaaatataa acttctgcca gaatacccag gtgttctctc tgatgtccag gaggagaaag 2400gcattaagta caaatttgaa gtatatgaga agaatgatta atcgatctta agtttaatct 2460ttcccggggg taccgtcgac tgcggccgcg aattccaagc ttgagtattc tatcgtgtca 2520cctaaataac ttggcgtaat catggtcata tctgtttcct gtgtgaaatt gttatccgct 2580cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 2640agtgagctaa ctcacattaa ttgcgttgcg cgatgcttcc attttgtgag ggttaatgct 2700tcgagaagac atgataagat acattgatga gtttggacaa accacaacaa gaatgcagtg 2760aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag 2820ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga 2880gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tccgataagg 2940atcgattccg gagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc 3000gggtgtggtg gttacgcgca cgtgaccgct acacttgcca gcgccctagc gcccgctcct 3060ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 3120cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 3180gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 3240acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 3300cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta 3360aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca 3420atttcgcctg tgtaccttct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg 3480gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 3540gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 3600gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 3660tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 3720ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 3780cctaggcttt tgcaaaaagc ttgattcttc tgacacaaca gtctcgaact taaggctaga 3840gccaccatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg 3900ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg 3960ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 4020gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca 4080gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg 4140gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat 4200gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa 4260catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg 4320gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg 4380cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg 4440gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat 4500caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac 4560cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc 4620cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc 4680ccaacctgcc atcacgatgg ccgcaataaa atatctttat tttcattaca tctgtgtgtt 4740ggttttttgt gtgaagatcc gcgtatggtg cactctcagt acaatctgct ctgatgccgc 4800atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 4860gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 4920gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 4980ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 5040tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 5100gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 5160acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 5220cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 5280catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 5340tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 5400cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 5460accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 5520cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 5580ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 5640accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 5700ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 5760attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 5820ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 5880tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 5940tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 6000gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 6060tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 6120ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 6180ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 6240agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 6300cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 6360caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 6420tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 6480ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 6540ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 6600gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 6660gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 6720tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 6780cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgg ctcgac 6836 194644 DNA Homo sapiens 19 gatcttcaat attggccatt agccatatta ttcattggttatatagcata aatcaatatt 60 ggctattggc cattgcatac gttgtatcta tatcataatatgtacattta tattggctca 120 tgtccaatat gaccgccatg ttggcattga ttattgactagttattaata gtaatcaatt 180 acggggtcat tagttcatag cccatatatg gagttccgcgttacataact tacggtaaat 240 ggcccgcctg gctgaccgcc caacgacccc cgcccattgacgtcaataat gacgtatgtt 300 cccatagtaa cgccaatagg gactttccat tgacgtcaatgggtggagta tttacggtaa 360 actgcccact tggcagtaca tcaagtgtat catatgccaagtccgccccc tattgacgtc 420 aatgacggta aatggcccgc ctggcattat gcccagtacatgaccttacg ggactttcct 480 acttggcagt acatctacgt attagtcatc gctattaccatggtgatgcg gttttggcag 540 tacaccaatg ggcgtggata gcggtttgac tcacggggatttccaagtct ccaccccatt 600 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacgggactttccaaa atgtcgtaac 660 aactgcgatc gcccgccccg ttgacgcaaa tgggcggtaggcgtgtacgg tgggaggtct 720 atataagcag agctcgttta gtgaaccgtc agatcactgaattctgacga cctactgatt 780 aacggccata gaggcctcct gcagatcact agaagctttattgcggtagt ttatcacagt 840 taaattgcta acgcagtcag tgcttctgac acaacagtctcgaacttaag ctgcagtgac 900 tctcttaaat ccaccatggc tacaggtgag tactcgctaccttaagagag gcctatctgg 960 ccagttagca gtcgaagaaa gaagtttaag agagccgaaacaagcgctca tgagcccgaa 1020 gtggcgagcc cgatcttccc catcggtgat gtcggcgatataggcgccag caaccgcacc 1080 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtagaggatccaca ggacgggtgt 1140 ggtcgccatg atcgcgtagt cgatagtggc tccaagtagcgaagcgagca ggactgggcg 1200 gcggccaaag cggtcggaca gtgctccgag aacgggtgcgcatagaaatt gcatcaacgc 1260 atatagcgct agatccttgc tagagtcgag atctgtcgagccatgtgagc aaaaggccag 1320 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgtttttccatag gctccgcccc 1380 cctgacgagc atcacaaaaa tcgacgctca agtcagaggtggcgaaaccc gacaggacta 1440 taaagatacc aggcgtttcc ccctggaagc tccctcgtgcgctctcctgt tccgaccctg 1500 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaagcgtggcgct ttctcatagc 1560 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgctccaagctggg ctgtgtgcac 1620 gaaccccccg ttcagcccga ccgctgcgcc ttatccggtaactatcgtct tgagtccaac 1680 ccggtaagac acgacttatc gccactggca gcagccactggtaacaggat tagcagagcg 1740 aggtatgtag gcggtgctac agagttcttg aagtggtggcctaactacgg ctacactaga 1800 aggacagtat ttggtatctg cgctctgctg aagccagttaccttcggaaa aagagttggt 1860 agctcttgat ccggcaaaca aaccaccgct ggtagcggtggtttttttgt ttgcaagcag 1920 cagattacgc gcagaaaaaa aggatctcaa gaagatcctttgatcttttc tacggggtct 1980 gacgctcagt ggaacgaaaa ctcacgttaa gggattttggtcatgagatt atcaaaaagg 2040 atcttcacct agatcctttt atcggtgtga aataccgcacagatgcgtaa ggagaaaata 2100 ccgcatcagg aaattgtaag cgttaataat tcagaagaactcgtcaagaa ggcgatagaa 2160 ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagcacgaggaagc ggtcagccca 2220 ttcgccgcca agctcttcag caatatcacg ggtagccaacgctatgtcct gatagcggtc 2280 cgccacaccc agccggccac agtcgatgaa tccagaaaagcggccatttt ccaccatgat 2340 attcggcaag caggcatcgc catgggtcac gacgagatcctcgccgtcgg gcatgctcgc 2400 cttgagcctg gcgaacagtt cggctggcgc gagcccctgatgctcttcgt ccagatcatc 2460 ctgatcgaca agaccggctt ccatccgagt acgtgctcgctcgatgcgat gtttcgcttg 2520 gtggtcgaat gggcaggtag ccggatcaag cgtatgcagccgccgcattg catcagccat 2580 gatggatact ttctcggcag gagcaaggtg agatgacaggagatcctgcc ccggcacttc 2640 gcccaatagc agccagtccc ttcccgcttc agtgacaacgtcgagcacag ctgcgcaagg 2700 aacgcccgtc gtggccagcc acgatagccg cgctgcctcgtcttgcagtt cattcagggc 2760 accggacagg tcggtcttga caaaaagaac cgggcgcccctgcgctgaca gccggaacac 2820 ggcggcatca gagcagccga ttgtctgttg tgcccagtcatagccgaata gcctctccac 2880 ccaagcggcc ggagaacctg cgtgcaatcc atcttgttcaatcatgcgaa acgatcctca 2940 tcctgtctct tgatcagagc ttgatcccct gcgccatcagatccttggcg gcgagaaagc 3000 catccagttt actttgcagg gcttgtcaac cttaccagataaaagtgctc atcattggaa 3060 aacgttcaat tctgaggcgg aaagaaccag ctgtggaatgtgtgtcagtt agggtgtgga 3120 aagtccccag gctccccagc aggcagaagt atgcaaagcatgcatctcaa ttagtcagca 3180 accaggtgtg gaaagtcccc aggctcccca gcaggcagaagtatgcaaag catgcatctc 3240 aattagtcag caaccatagt cccgccccta actccgcccatcccgcccct aactccgccc 3300 agttccgccc attctccgcc ccatggctga ctaattttttttatttatgc agaggccgag 3360 gccgcctcgg cctctgagct attccagaag tagtgaggaggcttttttgg aggcctaggc 3420 ttttgcaaaa agcttgattc ttctgacaca acagtctcgaacttaaggct agagccacca 3480 tgattgaaca agatggattg cacgcaggtt ctccggccgcttgggtggag aggctattcg 3540 gctatgactg ggcacaacag acaatcggct gctctgatgccgccgtgttc cggctgtcag 3600 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtccggtgccctg aatgaactgc 3660 aggacgaggc agcgcggcta tcgtggctgg ccacgacgggcgttccttgc gcagctgtgc 3720 tcgacgttgt cactgaagcg ggaagggact ggctgctattgggcgaagtg ccggggcagg 3780 atctcctgtc atctcacctt gctcctgccg agaaagtatccatcatggct gatgcaatgc 3840 ggcggctgca tacgcttgat ccggctacct gcccattcgaccaccaagcg aaacatcgca 3900 tcgagcgagc acgtactcgg atggaagccg gtcttgtcgatcaggatgat ctggacgaag 3960 agcatcaggg gctcgcgcca gccgaactgt tcgccaggctcaaggcgcgc atgcccgacg 4020 gcgaggatct cgtcgtgacc catggcgatg cctgcttgccgaatatcatg gtggaaaatg 4080 gccgcttttc tggattcatc gactgtggcc ggctgggtgtggcggaccgc tatcaggaca 4140 tagcgttggc tacccgtgat attgctgaag agcttggcggcgaatgggct gaccgcttcc 4200 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcatcgccttctat cgccttcttg 4260 acgagccatt ctgatggagg tagcggccgc taacctggttgctgactaat tgagatgcat 4320 gctttgcata cttctgcctg ctggggagcc tggggactttccacacccta actgacacac 4380 attccacagc tggttctttc cgcctcagaa ggtacacaggcgaaattgta agcgttaata 4440 ttttgttaaa attcgcgtta aatttttgtt aaatcagctcattttttaac caataggccg 4500 aaatcggcaa aatcccttat aaatcaaaag aatagaccgagatagggttg agtgttgttc 4560 cagtttggaa caagagtcca ctattaaaga acgtggactccaacgtcaaa gggcgaaaaa 4620 ccgtctatca gggcgatggc ccac 4644 20 5247 DNAHomo sapiens 20 gatcttcaat attggccatt agccatatta ttcattggtt atatagcataaatcaatatt 60 ggctattggc cattgcatac gttgtatcta tatcataata tgtacatttatattggctca 120 tgtccaatat gaccgccatg ttggcattga ttattgacta gttattaatagtaatcaatt 180 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataacttacggtaaat 240 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataatgacgtatgtt 300 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagtatttacggtaa 360 actgcccact tggcagtaca tcaagtgtat catatgccaa gtccgccccctattgacgtc 420 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttacgggactttcct 480 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcggttttggcag 540 tacaccaatg ggcgtggata gcggtttgac tcacggggat ttccaagtctccaccccatt 600 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaaatgtcgtaac 660 aactgcgatc gcccgccccg ttgacgcaaa tgggcggtag gcgtgtacggtgggaggtct 720 atataagcag agctcgttta gtgaaccgtc agatcactag aagctttattgcggtagttt 780 atcacagtta aattgctaac gcagtcagtg cttctgacac aacagtctcgaacttaagct 840 gcagtgactc tcttaaatcc accatggcta caggtgagta ctcgctaccttaagagaggc 900 ctatctggcc agttagcagt cgaagaaaga agtttaagag agccgaaacaagcgctcatg 960 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatataggcgccagca 1020 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagaggatccacagg 1080 acgggtgtgg tcgccatgat cgcgtagtcg atagtggctc caagtagcgaagcgagcagg 1140 actgggcggc ggccaaagcg gtcggacagt gctccgagaa cgggtgcgcatagaaattgc 1200 atcaacgcat atagcgctag atccttgcta gagtcgagat ctgtcgagccatgtgagcaa 1260 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgtttttccataggc 1320 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtggcgaaacccga 1380 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgctctcctgttc 1440 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagcgtggcgcttt 1500 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctccaagctgggct 1560 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaactatcgtcttg 1620 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggtaacaggatta 1680 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcctaactacggct 1740 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttaccttcggaaaaa 1800 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggtttttttgttt 1860 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttgatcttttcta 1920 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtcatgagattat 1980 caaaaaggat cttcacctag atccttttat cggtgtgaaa taccgcacagatgcgtaagg 2040 agaaaatacc gcatcaggaa attgtaagcg ttaataattc agaagaactcgtcaagaagg 2100 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcacgaggaagcgg 2160 tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgctatgtcctga 2220 tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcggccattttcc 2280 accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctcgccgtcgggc 2340 atgctcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatgctcttcgtcc 2400 agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctcgatgcgatgt 2460 ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccgccgcattgca 2520 tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggagatcctgcccc 2580 ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtcgagcacagct 2640 gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtcttgcagttca 2700 ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctgcgctgacagc 2760 cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcatagccgaatagc 2820 ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaatcatgcgaaac 2880 gatcctcatc ctgtctcttg atcagagctt gatcccctgc gccatcagatccttggcggc 2940 gagaaagcca tccagtttac tttgcagggc ttgtcaacct taccagataaaagtgctcat 3000 cattggaaaa cgttcaattc tgaggcggaa agaaccagct gtggaatgtgtgtcagttag 3060 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatgcatctcaatt 3120 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagtatgcaaagca 3180 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatcccgcccctaa 3240 ctccgcccag ttccgcccat tctccgcccc atggctgact aattttttttatttatgcag 3300 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggcttttttggag 3360 gcctaggctt ttgcaaaaag cttgattctt ctgacacaac agtctcgaacttaaggctag 3420 agccaccatg attgaacaag atggattgca cgcaggttct ccggccgcttgggtggagag 3480 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccgccgtgttccg 3540 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccggtgccctgaa 3600 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcgttccttgcgc 3660 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgggcgaagtgcc 3720 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatccatcatggctga 3780 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgaccaccaagcgaa 3840 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatcaggatgatct 3900 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctcaaggcgcgcat 3960 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccgaatatcatggt 4020 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtggcggaccgcta 4080 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcgaatgggctga 4140 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcgccttctatcg 4200 ccttcttgac gagccattct gctggatggc tacaggtcgc agccctggcgtcgtgattag 4260 tgatgatgaa ccaggttatg accttgattt attttgcata cctaatcattatgctgagga 4320 tttggaaagg gtgtttattc ctcatggact aattatggac aggactgaacgtcttgctcg 4380 agatgtgatg aaggagatgg gaggccatca cattgtagcc ctctgtgtgctcaagggggg 4440 ctataaattc tttgctgacc tgctggatta catcaaagca ctgaatagaaatagtgatag 4500 atccattcct atgactgtag attttatcag actgaagagc tattgtaatgaccagtcaac 4560 aggggacata aaagtaattg gtggagatga tctctcaact ttaactggaaagaatgtctt 4620 gattgtggaa gatataattg acactggcaa aacaatgcag actttgctttccttggtcag 4680 gcagtataat ccaaagatgg tcaaggtcgc aagcttgctg gtgaaaaggaccccacgaag 4740 tgttggatat aagccagact ttgttggatt tgaaattcca gacaagtttgttgtaggata 4800 tgcccttgac tataatgaat acttcaggga tttgaatcat gtttgtgtcattagtgaaac 4860 tggaaaagca aaatacaaag cctaagcggc cgctaacctg gttgctgactaattgagatg 4920 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacaccctaactgaca 4980 cacattccac agctggttct ttccgcctca gaaggtacac aggcgaaattgtaagcgtta 5040 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcattttttaaccaatagg 5100 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagatagggttgagtgttg 5160 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtcaaagggcgaa 5220 aaaccgtcta tcagggcgat ggcccac 5247 21 5382 DNA Homosapiens modified_base (890) a, c, t, g, other or unknown 21 cacctaaattgtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 60 ctcattttttaaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 120 cgagatagggttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 180 ctccaacgtcaaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 240 accctaatcaagttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 300 gagcccccgatttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 360 gaaagcgaaaggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 420 caccacacccgccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct 480 gcgcaactgttgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 540 agggggatgtgctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 600 ttgtaaaacgacggccagtg aattgtaata cgactcacta tagggcgaat tgggtacaat 660 tcaattcgtcgacctcgaaa ttctaccggg taggggaggc gcttttccca aggcagtctg 720 gagcatgcgctttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc 780 gcacacattccacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg 840 cgccaccttctactcctccc ctagtcagga agttcccccc cgccccgcan ctcgcgtcgt 900 gcaggacgtgacaaatggaa atagcacgtc tcactagtct cgtgcagatg gacaagcacc 960 gctgagcaatggagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg 1020 ctttctgggctcagaggctg gnaaggggtg ggtccggggg cgggctcagg ggcgggctca 1080 ggggcggggcgggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc 1140 gcacgtctgccgcgctgttc tcctcttcct catctccggg cctttcgacc tgcatccatc 1200 tagatctcgagcagctgaag cttaccatga ccgagtacaa gcccacggtg cgcctcgcca 1260 cccgcgacgacgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg 1320 ccacgcgccacaccgtcgac ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac 1380 tcttcctcacgcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg 1440 cggtggcggtctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg 1500 gcccgcgcatggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc 1560 tcctggcgccgcaccgggcc caaggagccc gcgtggttcc ttggcccacc gtcgggcgtc 1620 ttcgcccgaccaccagggca agggtctggc aagcgccgtc gtgctccccg gagtggaggc 1680 ggccgagcgcgccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt 1740 ctacgagcggctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac 1800 ctggtgcatgacccgcaagc ccggtgcctg acgcccgccc cacgacccgc agcgcccgac 1860 cgaaaggagcgcacgacccc atgcatcgat ggcactgggc aggtaagtat caaggttagc 1920 gatcttcaatattggccatt agccatatta ttcattggtt atatagcata aatcaatatt 1980 ggctattggccattgcatac gttgtatcta tatcataata tgtacattta tattggctca 2040 tgtccaatatgaccgccatg ttggcattga ttattgacta gttattaata gtaatcaatt 2100 acggggtcattagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 2160 ggcccgcctggctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 2220 cccatagtaacgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 2280 actgcccacttggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc 2340 aatgacggtaaatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct 2400 acttggcagtacatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag 2460 tacaccaatgggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt 2520 gacgtcaatgggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac 2580 aactgcgatcgcccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 2640 atataagcagagctcgttta gtgaaccgtc agatcactag aagctttatt gcggtagttt 2700 atcacagttaaattgctaac gcagtcagtg cttctgacac aacagtctcg aacttaagct 2760 gcagtgactctcttaattaa ccaccgctac aggtgagtac tcggatctgc taccttaaga 2820 gaggcctatctggccagtta gcagtcgaag aaagaagttt aagagagccg aaacaagcgc 2880 tcatgagcccgaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc 2940 cagcaaccgcacctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcc 3000 acaggacgggtgtggtcgcc atgatcgcgt agtcgatagt ggctccaagt agcgaagcga 3060 gcaggactgggcggcggcca aagcggtcgg acagtgctcc gagaacgggt gcgcatagaa 3120 attgcatcaacgcatatagc gctagatcct tgctagagtc gaggccgcca ccgcggtgga 3180 gctccagcttttgttccctt tagtgagggt taatttcgag cttggcgtaa tcatggtcat 3240 agctgtttcctgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 3300 gcataaagtgtaaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 3360 gctcactgcccgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 3420 aacgcgcggggagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 3480 cgctgcgctcggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 3540 ggttatccacagaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 3600 aggccaggaaccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 3660 acgagcatcacaaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 3720 gataccaggcgtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 3780 ttaccggatacctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 3840 gctgtaggtatctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 3900 cccccgttcagcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 3960 taagacacgacttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 4020 atgtaggcggtgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga 4080 cagtatttggtatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 4140 cttgatccggcaaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 4200 ttacgcgcagaaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 4260 ctcagtggaacgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 4320 tcacctagatccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 4380 aaacttggtctgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 4440 tatttcgttcatccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 4500 gcttaccatctggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 4560 atttatcagcaataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 4620 tatccgcctccatccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 4680 ttaatagtttgcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 4740 ttggtatggcttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 4800 tgttgtgcaaaaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 4860 ccgcagtgttatcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 4920 ccgtaagatgcttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 4980 tgcggcgaccgagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 5040 gaactttaaaagtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 5100 taccgctgttgagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 5160 cttttactttcaccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 5220 agggaataagggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 5280 gaagcatttatcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 5340 ataaacaaataggggttccg cgcacatttc cccgaaaagt gc 5382 22 9737 DNA Homo sapiensmodified_base (8347) a, c, t, g, other or unknown 22 gatcttcaatattggccatt agccatatta ttcattggtt atatagcata aatcaatatt 60 ggctattggccattgcatac gttgtatcta tatcataata tgtacattta tattggctca 120 tgtccaatatgaccgccatg ttggcattga ttattgacta gttattaata gtaatcaatt 180 acggggtcattagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 240 ggcccgcctggctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 300 cccatagtaacgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 360 actgcccacttggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc 420 aatgacggtaaatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct 480 acttggcagtacatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag 540 tacaccaatgggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt 600 gacgtcaatgggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac 660 aactgcgatcgcccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 720 atataagcagagctcgttta gtgaaccgtc agatcactga attctgacga cctactgatt 780 aacggccatagaggcctcct gcagaactgt cttagtgaca actatcgatt tccacacatt 840 atacgagccgatgttaattg tcaacagctc atgcatgacg tcccgggagc agacaagccc 900 gaccatggctcgagtaatac gactcactat agggcgacag gtgagtactc gctaccttaa 960 ggcctatctggccgtttaaa cagatgtgta taagagacag ctctcttaag gtagcctgtc 1020 tcttatacacatctagatcc ttgctagagt cgaccaattc tcatgtttga cagcttatca 1080 tcgcagatcctgagcttgta tggtgcactc tcagtacaat ctgctctgct gccgcatagt 1140 taagccagtatctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa 1200 tttaagctacaacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta 1260 ggcgttttgcgctgcttcgc gatgtacggg ccagatatac gcgtatctga ggggactagg 1320 gtgtgtttaggcgcccagcg gggcttcggt tgtacgcggt taggagtccc ctcaggatat 1380 agtagtttcgcttttgcata gggaggggga aatgtagtct tatgcaatac acttgtagtc 1440 ttgcaacatggtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc 1500 atgccgattggtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacagg 1560 tctgacatggattggacgaa ccactgaatt ccgcattgca gagataattg tatttaagtg 1620 cctagctcgatacaataaac gccatttgac cattcaccac attggtgtgc acctccaagc 1680 tgggtaccagctgctagcct cgagacgcgt gatttccttc gaagcttgtc atggttggtt 1740 cgctaaactgcatcgtcgct gtgtcccaga acatgggcat cggcaagaac ggggacctgc 1800 cctggccaccgctcaggaat gaattcagat atttccagag aatgaccaca acctcttcag 1860 tagaaggtaaacagaatctg gtgattatgg gtaagaagac ctggttctcc attcctgaga 1920 agaatcgacctttaaagggt agaattaatt tagttctcag cagagaactc aaggaacctc 1980 cacaaggagctcattttctt tccagaagtc tagatgatgc cttaaaactt actgaacaac 2040 cagaattagcaaataaagta gacatggtct ggatagttgg tggcagttct gtttataagg 2100 aagccatgaatcacccaggc catcttaaac tatttgtgac aaggatcatg caagactttg 2160 aaagtgacacgttttttcca gaaattgatt tggagaaata taaacttctg ccagaatacc 2220 caggtgttctctctgatgtc caggaggaga aaggcattaa gtacaaattt gaagtatatg 2280 agaagaatgttaattaaggg caccaataac tgccttaaaa aaattacgcc ccgccctgcc 2340 actcatcgcagtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaga 2400 cggcatgatgaacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt 2460 tgcccatggtgaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac 2520 tggtgaaactcacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag 2580 ggaaataggccaggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact 2640 gccggaaatcgtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga 2700 aaacggtgtaacaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca 2760 tacggaattccggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa 2820 acttgtgcttatttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct 2880 ggttataggtacattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt 2940 gggatatatcaacggtggta tatccagtga tttttttctc cattttagct tccttagctc 3000 ctgaaaatctcgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa 3060 agttggaacctcttacgtgc cgatcaacgt ctcattttcg ccaaattaat taaggcgcgc 3120 cgctctcctggctaggagtc acgtagaaag gactaccgac gaaggaactt gggtcgccgg 3180 tgtgttcgtatatggaggta gtaagacctc cctttacaac ctaaggcgag gaactgccct 3240 tgctattccacaatgtcgtc ttacaccatt gagtcgtctc ccctttggaa tggcccctgg 3300 acccggcccacaacctggcc cgctaaggga gtccattgtc tgttatttca tggtcttttt 3360 acaaactcatatatttgctg aggttttgaa ggatgcgatt aaggaccttg ttatgacaaa 3420 gcccgctcctacctgcaata tcagggtgac tgtgtgcagc tttgacgatg gagtagattt 3480 gcctccctggtttccaccta tggtggaagg ggctgccgcg gagggtgatg acggagatga 3540 cggagatgaaggaggtgatg gagatgaggg tgaggaaggg caggagtgat gtaacttgtt 3600 aggagacgccctcaatcgta ttaaaagccg tgtattcccc cgcactaaag aataaatccc 3660 cagtagacatcatgcgtgct gttggtgtat ttctggccat ctgtcttgtc accattttcg 3720 tcctcccaacatggggcaat tgggcatacc catgttgtca cgtcactcag ctccgcgctc 3780 aacaccttctcgcgttggaa aacattagcg acatttacct ggtgagcaat cagacatgcg 3840 acggctttagcctggcctcc ttaaattcac ctaagaatgg gagcaaccag catgcaggaa 3900 aaggacaagcagcgaaaatt cacgccccct tgggaggtgg cggcatatgc aaaggatagc 3960 actcccactctactactggg tatcatatgc tgactgtata tgcatgagga tagcatatgc 4020 tacccggatacagattagga tagcatatac tacccagata tagattagga tagcatatgc 4080 tacccagatatagattagga tagcctatgc tacccagata taaattagga tagcatatac 4140 tacccagatatagattagga tagcatatgc tacccagata tagattagga tagcctatgc 4200 tacccagatatagattagga tagcatatgc tacccagata tagattagga tagcatatgc 4260 tatccagatatttgggtagt atatgctacc cagatataaa ttaggatagc atatactacc 4320 ctaatctctattaggatagc atatgctacc cggatacaga ttaggatagc atatactacc 4380 cagatatagattaggatagc atatgctacc cagatataga ttaggatagc ctatgctacc 4440 cagatataaattaggatagc atatactacc cagatataga ttaggatagc atatgctacc 4500 cagatatagattaggatagc ctatgctacc cagatataga ttaggatagc atatgctatc 4560 cagatatttgggtagtatat gctacccatg gcaacattag cccaccgtgc tctcagcgac 4620 ctcgtgaatatgaggaccaa caaccctgtg cttggcgctc aggcgcaagt gtgtgtaatt 4680 tgtcctccagatcgcagcaa tcgcgcccct atcttggccc gcccacctac ttatgcaggt 4740 attccccggggtgccattag tggttttgtg ggcaagtggt ttgaccgcag tggttagcgg 4800 ggttacaatcagccaagtta ttacaccctt attttacagt ccaaaaccgc agggcggcgt 4860 gtgggggctgacgcgtgccc ccactccaca atttcaaaaa aaagagtggc cacttgtctt 4920 tgtttatgggccccattggc gtggagcccc gtttaatttt cgggggtgtt agagacaacc 4980 agtggagtccgctgctgtcg gcgtccactc tctttcccct tgttacaaat agagtgtaac 5040 aacatggttcacctgtcttg gtccctgcct gggacacatc ttaataaccc cagtatcata 5100 ttgcactaggattatgtgtt gcccatagcc ataaattcgt gtgagatgga catccagtct 5160 ttacggcttgtccccacccc atggatttct attgttaaag atattcagaa tgtttcattc 5220 ctacactagtatttattgcc caaggggttt gtgagggtta tattggtgtc atagcacaat 5280 gccaccactgaaccccccgt ccaaatttta ttctgggggc gtcacctgaa accttgtttt 5340 cgagcacctcacatacacct tactgttcac aactcagcag ttattctatt agctaaacga 5400 aggagaatgaagaagcaggc gaagattcag gagagttcac tgcccgctcc ttgatcttca 5460 gccactgcccttgtgactaa aatggttcac taccctcgtg gaatcctgac cccatgtaaa 5520 taaaaccgtgacagctcatg gggtgggaga tatcgctgtt ccttaggacc cttttactaa 5580 ccctaattcgatagcatatg cttcccgttg ggtaacatat gctattgaat tagggttagt 5640 ctggatagtatatactacta cccgggaagc atatgctacc cgtttagggt taacaagggg 5700 gccttataaacactattgct aatgccctct tgagggtccg cttatcggta gctacacagg 5760 cccctctgattgacgttggt gtagcctccc gtagtcttcc tgggcccctg ggaggtacat 5820 gtcccccagcattggtgtaa gagcttcagc caagagttac acataaaggc aatgttgtgt 5880 tgcagtccacagactgcaaa gtctgctcca ggatgaaagc cactcagtgt tggcaaatgt 5940 gcacatccatttataaggat gtcaactaca gtcagagaac ccctttgtgt ttggtccccc 6000 cccgtgtcacatgtggaaca gggcccagtt ggcaagttgt accaaccaac tgaagggatt 6060 acatgcactgccccgaatac aaaacaaaag cgctcctcgt accagcgaag aaggggcaga 6120 gatgccgtagtcaggtttag ttcgtccggc ggcgggcggc cgcaaggcgc gccggatcca 6180 caggacgggtgtggtcgcca tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag 6240 caggactgggcggcggccaa agcggtcgga cagtgctccg agaacgggtg cgcatagaaa 6300 ttgcatcaacgcatatagcg ctagatcctt gctagagtcg agatctgtcg agccatgtga 6360 gcaaaaggccagcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 6420 aggctccgcccccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 6480 ccgacaggactataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 6540 gttccgaccctgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 6600 ctttctcatagctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 6660 ggctgtgtgcacgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 6720 cttgagtccaacccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 6780 attagcagagcgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 6840 ggctacactagaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 6900 aaaagagttggtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 6960 gtttgcaagcagcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 7020 tctacggggtctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 7080 ttatcaaaaaggatcttcac ctagatcctt ttatcggtgt gaaataccgc acagatgcgt 7140 aaggagaaaataccgcatca ggaaattgta agcgttaata attcagaaga actcgtcaag 7200 aaggcgatagaaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa 7260 gcggtcagcccattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc 7320 ctgatagcggtccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt 7380 ttccaccatgatattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc 7440 gggcatgctcgccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc 7500 gtccagatcatcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg 7560 atgtttcgcttggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat 7620 tgcatcagccatgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg 7680 ccccggcacttcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac 7740 agctgcgcaaggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcttgcag 7800 ttcattcagggcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga 7860 cagccggaacacggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa 7920 tagcctctccacccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg 7980 aaacgatcctcatcctgtct cttgatcaga gcttgatccc ctgcgccatc agatccttgg 8040 cggcgagaaagccatccagt ttactttgca gggcttgtca accttaccag ataaaagtgc 8100 tcatcattggaaaacattca attcgtcgac ctcgaaattc taccgggtag gggaggcgct 8160 tttcccaaggcagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac 8220 aagtggcctctggcctcgca cacattccac atccaccggt aggcgccaac cggctccgtt 8280 ctttggtggccccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc 8340 cccgcanctcgcgtcgtgca ggacgtgaca aatggaaata gcacgtctca ctagtctcgt 8400 gcagatggacaagcaccgct gagcaatgga gcgggtaggc ctttggggca gcggccaata 8460 gcagctttgctccttcgctt tctgggctca gaggctggna aggggtgggt ccgggggcgg 8520 gctcaggggcgggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt 8580 ctgcacgcttcaaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct 8640 ttcgacctgcatccatctag atctcgagca gctgaagctt accatgaccg agtacaagcc 8700 cacggtgcgcctcgccaccc gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc 8760 gttcgccgactaccccgcca cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt 8820 caccgagctgcaagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt 8880 cgcggacgacggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc 8940 ggtgttcgccgagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca 9000 gcaacagatggaaggcctcc tggcgccgca ccgggcccaa ggagcccgcg tggttccttg 9060 gcccaccgtcgggcgtcttc gcccgaccac cagggcaagg gtctggcaag cgccgtcgtg 9120 ctccccggagtggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg 9180 ccccgcaacctccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg 9240 cccgaaggaccgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac 9300 gacccgcagcgcccgaccga aaggagcgca cgaccccatg catcgatggc actgggcagg 9360 taagtatcaaggttagcggc cgctaacctg gttgctgact aattgagatg catgctttgc 9420 atacttctgcctgctgggga gcctggggac tttccacacc ctaactgaca cacattccac 9480 agctggttctttccgcctca gaaggtacac aggcgaaatt gtaagcgtta atattttgtt 9540 aaaattcgcgttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg 9600 caaaatcccttataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg 9660 gaacaagagtccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta 9720 tcagggcgatggcccac 9737 23 9737 DNA Homo sapiens modified_base (8347) a, c, t, g,other or unknown 23 gatcttcaat attggccatt agccatatta ttcattggttatatagcata aatcaatatt 60 ggctattggc cattgcatac gttgtatcta tatcataatatgtacattta tattggctca 120 tgtccaatat gaccgccatg ttggcattga ttattgactagttattaata gtaatcaatt 180 acggggtcat tagttcatag cccatatatg gagttccgcgttacataact tacggtaaat 240 ggcccgcctg gctgaccgcc caacgacccc cgcccattgacgtcaataat gacgtatgtt 300 cccatagtaa cgccaatagg gactttccat tgacgtcaatgggtggagta tttacggtaa 360 actgcccact tggcagtaca tcaagtgtat catatgccaagtccgccccc tattgacgtc 420 aatgacggta aatggcccgc ctggcattat gcccagtacatgaccttacg ggactttcct 480 acttggcagt acatctacgt attagtcatc gctattaccatggtgatgcg gttttggcag 540 tacaccaatg ggcgtggata gcggtttgac tcacggggatttccaagtct ccaccccatt 600 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacgggactttccaaa atgtcgtaac 660 aactgcgatc gcccgccccg ttgacgcaaa tgggcggtaggcgtgtacgg tgggaggtct 720 atataagcag agctcgttta gtgaaccgtc agatcactgaattctgacga cctactgatt 780 aacggccata gaggcctcct gcagaactgt cttagtgacaactatcgatt tccacacatt 840 atacgagccg atgttaattg tcaacagctc atgcatgacgtcccgggagc agacaagccc 900 gaccatggct cgagtaatac gactcactat agggcgacaggtgagtactc gctaccttaa 960 ggcctatctg gccgtttaaa cagatgtgta taagagacagctctcttaag gtagcctgtc 1020 tcttatacac atctagatcc ttgctagagt cgaccaattctcatgtttga cagcttatca 1080 tcgcagatcc tgagcttgta tggtgcactc tcagtacaatctgctctgct gccgcatagt 1140 taagccagta tctgctccct gcttgtgtgt tggaggtcgctgagtagtgc gcgagcaaaa 1200 tttaagctac aacaaggcaa ggcttgaccg acaattgcatgaagaatctg cttagggtta 1260 ggcgttttgc gctgcttcgc gatgtacggg ccagatatacgcgtatctga ggggactagg 1320 gtgtgtttag gcgcccagcg gggcttcggt tgtacgcggttaggagtccc ctcaggatat 1380 agtagtttcg cttttgcata gggaggggga aatgtagtcttatgcaatac acttgtagtc 1440 ttgcaacatg gtaacgatga gttagcaaca tgccttacaaggagagaaaa agcaccgtgc 1500 atgccgattg gtggaagtaa ggtggtacga tcgtgccttattaggaaggc aacagacagg 1560 tctgacatgg attggacgaa ccactgaatt ccgcattgcagagataattg tatttaagtg 1620 cctagctcga tacaataaac gccatttgac cattcaccacattggtgtgc acctccaagc 1680 tgggtaccag ctgctagcct cgagacgcgt gatttccttcgaagcttgtc atggttggtt 1740 cgctaaactg catcgtcgct gtgtcccaga acatgggcatcggcaagaac ggggacctgc 1800 cctggccacc gctcaggaat gaattcagat atttccagagaatgaccaca acctcttcag 1860 tagaaggtaa acagaatctg gtgattatgg gtaagaagacctggttctcc attcctgaga 1920 agaatcgacc tttaaagggt agaattaatt tagttctcagcagagaactc aaggaacctc 1980 cacaaggagc tcattttctt tccagaagtc tagatgatgccttaaaactt actgaacaac 2040 cagaattagc aaataaagta gacatggtct ggatagttggtggcagttct gtttataagg 2100 aagccatgaa tcacccaggc catcttaaac tatttgtgacaaggatcatg caagactttg 2160 aaagtgacac gttttttcca gaaattgatt tggagaaatataaacttctg ccagaatacc 2220 caggtgttct ctctgatgtc caggaggaga aaggcattaagtacaaattt gaagtatatg 2280 agaagaatgt taattaaggg caccaataac tgccttaaaaaaattacgcc ccgccctgcc 2340 actcatcgca gtactgttgt aattcattaa gcattctgccgacatggaag ccatcacaga 2400 cggcatgatg aacctgaatc gccagcggca tcagcaccttgtcgccttgc gtataatatt 2460 tgcccatggt gaaaacgggg gcgaagaagt tgtccatattggccacgttt aaatcaaaac 2520 tggtgaaact cacccaggga ttggctgaga cgaaaaacatattctcaata aaccctttag 2580 ggaaataggc caggttttca ccgtaacacg ccacatcttgcgaatatatg tgtagaaact 2640 gccggaaatc gtcgtggtat tcactccaga gcgatgaaaacgtttcagtt tgctcatgga 2700 aaacggtgta acaagggtga acactatccc atatcaccagctcaccgtct ttcattgcca 2760 tacggaattc cggatgagca ttcatcaggc gggcaagaatgtgaataaag gccggataaa 2820 acttgtgctt atttttcttt acggtcttta aaaaggccgtaatatccagc tgaacggtct 2880 ggttataggt acattgagca actgactgaa atgcctcaaaatgttcttta cgatgccatt 2940 gggatatatc aacggtggta tatccagtga tttttttctccattttagct tccttagctc 3000 ctgaaaatct cgataactca aaaaatacgc ccggtagtgatcttatttca ttatggtgaa 3060 agttggaacc tcttacgtgc cgatcaacgt ctcattttcgccaaattaat taaggcgcgc 3120 cgctctcctg gctaggagtc acgtagaaag gactaccgacgaaggaactt gggtcgccgg 3180 tgtgttcgta tatggaggta gtaagacctc cctttacaacctaaggcgag gaactgccct 3240 tgctattcca caatgtcgtc ttacaccatt gagtcgtctcccctttggaa tggcccctgg 3300 acccggccca caacctggcc cgctaaggga gtccattgtctgttatttca tggtcttttt 3360 acaaactcat atatttgctg aggttttgaa ggatgcgattaaggaccttg ttatgacaaa 3420 gcccgctcct acctgcaata tcagggtgac tgtgtgcagctttgacgatg gagtagattt 3480 gcctccctgg tttccaccta tggtggaagg ggctgccgcggagggtgatg acggagatga 3540 cggagatgaa ggaggtgatg gagatgaggg tgaggaagggcaggagtgat gtaacttgtt 3600 aggagacgcc ctcaatcgta ttaaaagccg tgtattcccccgcactaaag aataaatccc 3660 cagtagacat catgcgtgct gttggtgtat ttctggccatctgtcttgtc accattttcg 3720 tcctcccaac atggggcaat tgggcatacc catgttgtcacgtcactcag ctccgcgctc 3780 aacaccttct cgcgttggaa aacattagcg acatttacctggtgagcaat cagacatgcg 3840 acggctttag cctggcctcc ttaaattcac ctaagaatgggagcaaccag catgcaggaa 3900 aaggacaagc agcgaaaatt cacgccccct tgggaggtggcggcatatgc aaaggatagc 3960 actcccactc tactactggg tatcatatgc tgactgtatatgcatgagga tagcatatgc 4020 tacccggata cagattagga tagcatatac tacccagatatagattagga tagcatatgc 4080 tacccagata tagattagga tagcctatgc tacccagatataaattagga tagcatatac 4140 tacccagata tagattagga tagcatatgc tacccagatatagattagga tagcctatgc 4200 tacccagata tagattagga tagcatatgc tacccagatatagattagga tagcatatgc 4260 tatccagata tttgggtagt atatgctacc cagatataaattaggatagc atatactacc 4320 ctaatctcta ttaggatagc atatgctacc cggatacagattaggatagc atatactacc 4380 cagatataga ttaggatagc atatgctacc cagatatagattaggatagc ctatgctacc 4440 cagatataaa ttaggatagc atatactacc cagatatagattaggatagc atatgctacc 4500 cagatataga ttaggatagc ctatgctacc cagatatagattaggatagc atatgctatc 4560 cagatatttg ggtagtatat gctacccatg gcaacattagcccaccgtgc tctcagcgac 4620 ctcgtgaata tgaggaccaa caaccctgtg cttggcgctcaggcgcaagt gtgtgtaatt 4680 tgtcctccag atcgcagcaa tcgcgcccct atcttggcccgcccacctac ttatgcaggt 4740 attccccggg gtgccattag tggttttgtg ggcaagtggtttgaccgcag tggttagcgg 4800 ggttacaatc agccaagtta ttacaccctt attttacagtccaaaaccgc agggcggcgt 4860 gtgggggctg acgcgtgccc ccactccaca atttcaaaaaaaagagtggc cacttgtctt 4920 tgtttatggg ccccattggc gtggagcccc gtttaattttcgggggtgtt agagacaacc 4980 agtggagtcc gctgctgtcg gcgtccactc tctttccccttgttacaaat agagtgtaac 5040 aacatggttc acctgtcttg gtccctgcct gggacacatcttaataaccc cagtatcata 5100 ttgcactagg attatgtgtt gcccatagcc ataaattcgtgtgagatgga catccagtct 5160 ttacggcttg tccccacccc atggatttct attgttaaagatattcagaa tgtttcattc 5220 ctacactagt atttattgcc caaggggttt gtgagggttatattggtgtc atagcacaat 5280 gccaccactg aaccccccgt ccaaatttta ttctgggggcgtcacctgaa accttgtttt 5340 cgagcacctc acatacacct tactgttcac aactcagcagttattctatt agctaaacga 5400 aggagaatga agaagcaggc gaagattcag gagagttcactgcccgctcc ttgatcttca 5460 gccactgccc ttgtgactaa aatggttcac taccctcgtggaatcctgac cccatgtaaa 5520 taaaaccgtg acagctcatg gggtgggaga tatcgctgttccttaggacc cttttactaa 5580 ccctaattcg atagcatatg cttcccgttg ggtaacatatgctattgaat tagggttagt 5640 ctggatagta tatactacta cccgggaagc atatgctacccgtttagggt taacaagggg 5700 gccttataaa cactattgct aatgccctct tgagggtccgcttatcggta gctacacagg 5760 cccctctgat tgacgttggt gtagcctccc gtagtcttcctgggcccctg ggaggtacat 5820 gtcccccagc attggtgtaa gagcttcagc caagagttacacataaaggc aatgttgtgt 5880 tgcagtccac agactgcaaa gtctgctcca ggatgaaagccactcagtgt tggcaaatgt 5940 gcacatccat ttataaggat gtcaactaca gtcagagaacccctttgtgt ttggtccccc 6000 cccgtgtcac atgtggaaca gggcccagtt ggcaagttgtaccaaccaac tgaagggatt 6060 acatgcactg ccccgaatac aaaacaaaag cgctcctcgtaccagcgaag aaggggcaga 6120 gatgccgtag tcaggtttag ttcgtccggc ggcgggcggccgcaaggcgc gccggatcca 6180 caggacgggt gtggtcgcca tgatcgcgta gtcgatagtggctccaagta gcgaagcgag 6240 caggactggg cggcggccaa agcggtcgga cagtgctccgagaacgggtg cgcatagaaa 6300 ttgcatcaac gcatatagcg ctagatcctt gctagagtcgagatctgtcg agccatgtga 6360 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccgcgttgctggc gtttttccat 6420 aggctccgcc cccctgacga gcatcacaaa aatcgacgctcaagtcagag gtggcgaaac 6480 ccgacaggac tataaagata ccaggcgttt ccccctggaagctccctcgt gcgctctcct 6540 gttccgaccc tgccgcttac cggatacctg tccgcctttctcccttcggg aagcgtggcg 6600 ctttctcata gctcacgctg taggtatctc agttcggtgtaggtcgttcg ctccaagctg 6660 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcgccttatccgg taactatcgt 6720 cttgagtcca acccggtaag acacgactta tcgccactggcagcagccac tggtaacagg 6780 attagcagag cgaggtatgt aggcggtgct acagagttcttgaagtggtg gcctaactac 6840 ggctacacta gaaggacagt atttggtatc tgcgctctgctgaagccagt taccttcgga 6900 aaaagagttg gtagctcttg atccggcaaa caaaccaccgctggtagcgg tggttttttt 6960 gtttgcaagc agcagattac gcgcagaaaa aaaggatctcaagaagatcc tttgatcttt 7020 tctacggggt ctgacgctca gtggaacgaa aactcacgttaagggatttt ggtcatgaga 7080 ttatcaaaaa ggatcttcac ctagatcctt ttatcggtgtgaaataccgc acagatgcgt 7140 aaggagaaaa taccgcatca ggaaattgta agcgttaataattcagaaga actcgtcaag 7200 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcgataccgtaaa gcacgaggaa 7260 gcggtcagcc cattcgccgc caagctcttc agcaatatcacgggtagcca acgctatgtc 7320 ctgatagcgg tccgccacac ccagccggcc acagtcgatgaatccagaaa agcggccatt 7380 ttccaccatg atattcggca agcaggcatc gccatgggtcacgacgagat cctcgccgtc 7440 gggcatgctc gccttgagcc tggcgaacag ttcggctggcgcgagcccct gatgctcttc 7500 gtccagatca tcctgatcga caagaccggc ttccatccgagtacgtgctc gctcgatgcg 7560 atgtttcgct tggtggtcga atgggcaggt agccggatcaagcgtatgca gccgccgcat 7620 tgcatcagcc atgatggata ctttctcggc aggagcaaggtgagatgaca ggagatcctg 7680 ccccggcact tcgcccaata gcagccagtc ccttcccgcttcagtgacaa cgtcgagcac 7740 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagccgcgctgcct cgtcttgcag 7800 ttcattcagg gcaccggaca ggtcggtctt gacaaaaagaaccgggcgcc cctgcgctga 7860 cagccggaac acggcggcat cagagcagcc gattgtctgttgtgcccagt catagccgaa 7920 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaatccatcttgtt caatcatgcg 7980 aaacgatcct catcctgtct cttgatcaga gcttgatcccctgcgccatc agatccttgg 8040 cggcgagaaa gccatccagt ttactttgca gggcttgtcaaccttaccag ataaaagtgc 8100 tcatcattgg aaaacattca attcgtcgac ctcgaaattctaccgggtag gggaggcgct 8160 tttcccaagg cagtctggag catgcgcttt agcagccccgctgggcactt ggcgctacac 8220 aagtggcctc tggcctcgca cacattccac atccaccggtaggcgccaac cggctccgtt 8280 ctttggtggc cccttcgcgc caccttctac tcctcccctagtcaggaagt tcccccccgc 8340 cccgcanctc gcgtcgtgca ggacgtgaca aatggaaatagcacgtctca ctagtctcgt 8400 gcagatggac aagcaccgct gagcaatgga gcgggtaggcctttggggca gcggccaata 8460 gcagctttgc tccttcgctt tctgggctca gaggctggnaaggggtgggt ccgggggcgg 8520 gctcaggggc gggctcaggg gcggggcggg cgcccgaaggtcctccggag gcccggcatt 8580 ctgcacgctt caaaagcgca cgtctgccgc gctgttctcctcttcctcat ctccgggcct 8640 ttcgacctgc atccatctag atctcgagca gctgaagcttaccatgaccg agtacaagcc 8700 cacggtgcgc ctcgccaccc gcgacgacgt cccccgggccgtacgcaccc tcgccgccgc 8760 gttcgccgac taccccgcca cgcgccacac cgtcgacccggaccgccaca tcgagcgggt 8820 caccgagctg caagaactct tcctcacgcg cgtcgggctcgacatcggca aggtgtgggt 8880 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccggagagcgtcg aagcgggggc 8940 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagcggttcccggc tggccgcgca 9000 gcaacagatg gaaggcctcc tggcgccgca ccgggcccaaggagcccgcg tggttccttg 9060 gcccaccgtc gggcgtcttc gcccgaccac cagggcaagggtctggcaag cgccgtcgtg 9120 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccgccttcctgga gacctccgcg 9180 ccccgcaacc tccccttcta cgagcggctc ggcttcaccgtcaccgccga cgtcgaggtg 9240 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccggtgcctgacg cccgccccac 9300 gacccgcagc gcccgaccga aaggagcgca cgaccccatgcatcgatggc actgggcagg 9360 taagtatcaa ggttagcggc cgctaacctg gttgctgactaattgagatg catgctttgc 9420 atacttctgc ctgctgggga gcctggggac tttccacaccctaactgaca cacattccac 9480 agctggttct ttccgcctca gaaggtacac aggcgaaattgtaagcgtta atattttgtt 9540 aaaattcgcg ttaaattttt gttaaatcag ctcattttttaaccaatagg ccgaaatcgg 9600 caaaatccct tataaatcaa aagaatagac cgagatagggttgagtgttg ttccagtttg 9660 gaacaagagt ccactattaa agaacgtgga ctccaacgtcaaagggcgaa aaaccgtcta 9720 tcagggcgat ggcccac 9737 24 9871 DNA Homosapiens modified_base (8481) a, c, t, g, other or unknown 24 gatcttcaatattggccatt agccatatta ttcattggtt atatagcata aatcaatatt 60 ggctattggccattgcatac gttgtatcta tatcataata tgtacattta tattggctca 120 tgtccaatatgaccgccatg ttggcattga ttattgacta gttattaata gtaatcaatt 180 acggggtcattagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 240 ggcccgcctggctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 300 cccatagtaacgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 360 actgcccacttggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc 420 aatgacggtaaatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct 480 acttggcagtacatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag 540 tacaccaatgggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt 600 gacgtcaatgggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac 660 aactgcgatcgcccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 720 atataagcagagctcgttta gtgaaccgtc agatcactga attctgacga cctactgatt 780 aaagatctaagctagcgccg ccaccatggg ccctaaaaag aagcgtaaag tcgccccccc 840 gaccgatgtcagcctggggg acgagctcca cttagacggc gaggacgtgg cgatggcgca 900 tgccgacgcgctagacgatt tcgatctgga catgttgggg gacggggatt ccccggggcc 960 gggatttaccccccacgact ccgcccccta cggcgctctg gatatggccg acttcgagtt 1020 tgagcagatgtttaccgatg cccttggaat tgacgagtac ggtggggaat tcaggtgagt 1080 actcgctaccttaaggccta tctggccgtt taaacagatg tgtataagag acagctctct 1140 taaggtagcctgtctcttat acacatctag atccttgcta gagtcgacca attctcatgt 1200 ttgacagcttatcatcgcag atcctgagct tgtatggtgc actctcagta caatctgctc 1260 tgctgccgcatagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta 1320 gtgcgcgagcaaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa 1380 tctgcttagggttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgtat 1440 ctgaggggactagggtgtgt ttaggcgccc agcggggctt cggttgtacg cggttaggag 1500 tcccctcaggatatagtagt ttcgcttttg catagggagg gggaaatgta gtcttatgca 1560 atacacttgtagtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag 1620 aaaaagcaccgtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga 1680 aggcaacagacaggtctgac atggattgga cgaaccactg aattccgcat tgcagagata 1740 attgtatttaagtgcctagc tcgatacaat aaacgccatt tgaccattca ccacattggt 1800 gtgcacctccaagctgggta ccagctgcta gcctcgagac gcgtgatttc cttcgaagct 1860 tgtcatggttggttcgctaa actgcatcgt cgctgtgtcc cagaacatgg gcatcggcaa 1920 gaacggggacctgccctggc caccgctcag gaatgaattc agatatttcc agagaatgac 1980 cacaacctcttcagtagaag gtaaacagaa tctggtgatt atgggtaaga agacctggtt 2040 ctccattcctgagaagaatc gacctttaaa gggtagaatt aatttagttc tcagcagaga 2100 actcaaggaacctccacaag gagctcattt tctttccaga agtctagatg atgccttaaa 2160 acttactgaacaaccagaat tagcaaataa agtagacatg gtctggatag ttggtggcag 2220 ttctgtttataaggaagcca tgaatcaccc aggccatctt aaactatttg tgacaaggat 2280 catgcaagactttgaaagtg acacgttttt tccagaaatt gatttggaga aatataaact 2340 tctgccagaatacccaggtg ttctctctga tgtccaggag gagaaaggca ttaagtacaa 2400 atttgaagtatatgagaaga atgttaatta agggcaccaa taactgcctt aaaaaaatta 2460 cgccccgccctgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg 2520 gaagccatcacagacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc 2580 ttgcgtataatatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac 2640 gtttaaatcaaaactggtga aactcaccca gggattggct gagacgaaaa acatattctc 2700 aataaaccctttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata 2760 tatgtgtagaaactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc 2820 agtttgctcatggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc 2880 gtctttcattgccatacgga attccggatg agcattcatc aggcgggcaa gaatgtgaat 2940 aaaggccggataaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc 3000 cagctgaacggtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc 3060 tttacgatgccattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt 3120 agcttccttagctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat 3180 ttcattatggtgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccaaat 3240 taattaaggcgcgccgctct cctggctagg agtcacgtag aaaggactac cgacgaagga 3300 acttgggtcgccggtgtgtt cgtatatgga ggtagtaaga cctcccttta caacctaagg 3360 cgaggaactgcccttgctat tccacaatgt cgtcttacac cattgagtcg tctccccttt 3420 ggaatggcccctggacccgg cccacaacct ggcccgctaa gggagtccat tgtctgttat 3480 ttcatggtctttttacaaac tcatatattt gctgaggttt tgaaggatgc gattaaggac 3540 cttgttatgacaaagcccgc tcctacctgc aatatcaggg tgactgtgtg cagctttgac 3600 gatggagtagatttgcctcc ctggtttcca cctatggtgg aaggggctgc cgcggagggt 3660 gatgacggagatgacggaga tgaaggaggt gatggagatg agggtgagga agggcaggag 3720 tgatgtaacttgttaggaga cgccctcaat cgtattaaaa gccgtgtatt cccccgcact 3780 aaagaataaatccccagtag acatcatgcg tgctgttggt gtatttctgg ccatctgtct 3840 tgtcaccattttcgtcctcc caacatgggg caattgggca tacccatgtt gtcacgtcac 3900 tcagctccgcgctcaacacc ttctcgcgtt ggaaaacatt agcgacattt acctggtgag 3960 caatcagacatgcgacggct ttagcctggc ctccttaaat tcacctaaga atgggagcaa 4020 ccagcatgcaggaaaaggac aagcagcgaa aattcacgcc cccttgggag gtggcggcat 4080 atgcaaaggatagcactccc actctactac tgggtatcat atgctgactg tatatgcatg 4140 aggatagcatatgctacccg gatacagatt aggatagcat atactaccca gatatagatt 4200 aggatagcatatgctaccca gatatagatt aggatagcct atgctaccca gatataaatt 4260 aggatagcatatactaccca gatatagatt aggatagcat atgctaccca gatatagatt 4320 aggatagcctatgctaccca gatatagatt aggatagcat atgctaccca gatatagatt 4380 aggatagcatatgctatcca gatatttggg tagtatatgc tacccagata taaattagga 4440 tagcatatactaccctaatc tctattagga tagcatatgc tacccggata cagattagga 4500 tagcatatactacccagata tagattagga tagcatatgc tacccagata tagattagga 4560 tagcctatgctacccagata taaattagga tagcatatac tacccagata tagattagga 4620 tagcatatgctacccagata tagattagga tagcctatgc tacccagata tagattagga 4680 tagcatatgctatccagata tttgggtagt atatgctacc catggcaaca ttagcccacc 4740 gtgctctcagcgacctcgtg aatatgagga ccaacaaccc tgtgcttggc gctcaggcgc 4800 aagtgtgtgtaatttgtcct ccagatcgca gcaatcgcgc ccctatcttg gcccgcccac 4860 ctacttatgcaggtattccc cggggtgcca ttagtggttt tgtgggcaag tggtttgacc 4920 gcagtggttagcggggttac aatcagccaa gttattacac ccttatttta cagtccaaaa 4980 ccgcagggcggcgtgtgggg gctgacgcgt gcccccactc cacaatttca aaaaaaagag 5040 tggccacttgtctttgttta tgggccccat tggcgtggag ccccgtttaa ttttcggggg 5100 tgttagagacaaccagtgga gtccgctgct gtcggcgtcc actctctttc cccttgttac 5160 aaatagagtgtaacaacatg gttcacctgt cttggtccct gcctgggaca catcttaata 5220 accccagtatcatattgcac taggattatg tgttgcccat agccataaat tcgtgtgaga 5280 tggacatccagtctttacgg cttgtcccca ccccatggat ttctattgtt aaagatattc 5340 agaatgtttcattcctacac tagtatttat tgcccaaggg gtttgtgagg gttatattgg 5400 tgtcatagcacaatgccacc actgaacccc ccgtccaaat tttattctgg gggcgtcacc 5460 tgaaaccttgttttcgagca cctcacatac accttactgt tcacaactca gcagttattc 5520 tattagctaaacgaaggaga atgaagaagc aggcgaagat tcaggagagt tcactgcccg 5580 ctccttgatcttcagccact gcccttgtga ctaaaatggt tcactaccct cgtggaatcc 5640 tgaccccatgtaaataaaac cgtgacagct catggggtgg gagatatcgc tgttccttag 5700 gacccttttactaaccctaa ttcgatagca tatgcttccc gttgggtaac atatgctatt 5760 gaattagggttagtctggat agtatatact actacccggg aagcatatgc tacccgttta 5820 gggttaacaagggggcctta taaacactat tgctaatgcc ctcttgaggg tccgcttatc 5880 ggtagctacacaggcccctc tgattgacgt tggtgtagcc tcccgtagtc ttcctgggcc 5940 cctgggaggtacatgtcccc cagcattggt gtaagagctt cagccaagag ttacacataa 6000 aggcaatgttgtgttgcagt ccacagactg caaagtctgc tccaggatga aagccactca 6060 gtgttggcaaatgtgcacat ccatttataa ggatgtcaac tacagtcaga gaaccccttt 6120 gtgtttggtccccccccgtg tcacatgtgg aacagggccc agttggcaag ttgtaccaac 6180 caactgaagggattacatgc actgccccga atacaaaaca aaagcgctcc tcgtaccagc 6240 gaagaaggggcagagatgcc gtagtcaggt ttagttcgtc cggcggcggg cggccgcaag 6300 gcgcgccggatccacaggac gggtgtggtc gccatgatcg cgtagtcgat agtggctcca 6360 agtagcgaagcgagcaggac tgggcggcgg ccaaagcggt cggacagtgc tccgagaacg 6420 ggtgcgcatagaaattgcat caacgcatat agcgctagat ccttgctaga gtcgagatct 6480 gtcgagccatgtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 6540 tggcgtttttccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 6600 agaggtggcgaaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 6660 tcgtgcgctctcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 6720 cgggaagcgtggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 6780 ttcgctccaagctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 6840 ccggtaactatcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 6900 ccactggtaacaggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 6960 ggtggcctaactacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 7020 cagttaccttcggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 7080 gcggtggtttttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 7140 atcctttgatcttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 7200 ttttggtcatgagattatca aaaaggatct tcacctagat ccttttatcg gtgtgaaata 7260 ccgcacagatgcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt aataattcag 7320 aagaactcgtcaagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg 7380 taaagcacgaggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta 7440 gccaacgctatgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca 7500 gaaaagcggccattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg 7560 agatcctcgccgtcgggcat gctcgccttg agcctggcga acagttcggc tggcgcgagc 7620 ccctgatgctcttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt 7680 gctcgctcgatgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta 7740 tgcagccgccgcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat 7800 gacaggagatcctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg 7860 acaacgtcgagcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct 7920 gcctcgtcttgcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg 7980 cgcccctgcgctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc 8040 cagtcatagccgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct 8100 tgttcaatcatgcgaaacga tcctcatcct gtctcttgat cagagcttga tcccctgcgc 8160 catcagatccttggcggcga gaaagccatc cagtttactt tgcagggctt gtcaacctta 8220 ccagataaaagtgctcatca ttggaaaaca ttcaattcgt cgacctcgaa attctaccgg 8280 gtaggggaggcgcttttccc aaggcagtct ggagcatgcg ctttagcagc cccgctgggc 8340 acttggcgctacacaagtgg cctctggcct cgcacacatt ccacatccac cggtaggcgc 8400 caaccggctccgttctttgg tggccccttc gcgccacctt ctactcctcc cctagtcagg 8460 aagttcccccccgccccgca nctcgcgtcg tgcaggacgt gacaaatgga aatagcacgt 8520 ctcactagtctcgtgcagat ggacaagcac cgctgagcaa tggagcgggt aggcctttgg 8580 ggcagcggccaatagcagct ttgctccttc gctttctggg ctcagaggct ggnaaggggt 8640 gggtccgggggcgggctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc 8700 ggaggcccggcattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc 8760 tcatctccgggcctttcgac ctgcatccat ctagatctcg agcagctgaa gcttaccatg 8820 accgagtacaagcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc 8880 accctcgccgccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc 8940 cacatcgagcgggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc 9000 ggcaaggtgtgggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc 9060 gtcgaagcgggggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc 9120 cggctggccgcgcagcaaca gatggaaggc ctcctggcgc cgcaccgggc ccaaggagcc 9180 cgcgtggttccttggcccac cgtcgggcgt cttcgcccga ccaccagggc aagggtctgg 9240 caagcgccgtcgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc 9300 tggagacctccgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg 9360 ccgacgtcgaggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct 9420 gacgcccgccccacgacccg cagcgcccga ccgaaaggag cgcacgaccc catgcatcga 9480 tggcactgggcaggtaagta tcaaggttag cggccgctaa cctggttgct gactaattga 9540 gatgcatgctttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact 9600 gacacacattccacagctgg ttctttccgc ctcagaaggt acacaggcga aattgtaagc 9660 gttaatattttgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa 9720 taggccgaaatcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt 9780 gttgttccagtttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg 9840 cgaaaaaccgtctatcaggg cgatggccca c 9871 25 10060 DNA Homo sapiens modified_base(8670) a, c, t, g, other or unknown 25 gatcttcaat attggccatt agccatattattcattggtt atatagcata aatcaatatt 60 ggctattggc cattgcatac gttgtatctatatcataata tgtacattta tattggctca 120 tgtccaatat gaccgccatg ttggcattgattattgacta gttattaata gtaatcaatt 180 acggggtcat tagttcatag cccatatatggagttccgcg ttacataact tacggtaaat 240 ggcccgcctg gctgaccgcc caacgacccccgcccattga cgtcaataat gacgtatgtt 300 cccatagtaa cgccaatagg gactttccattgacgtcaat gggtggagta tttacggtaa 360 actgcccact tggcagtaca tcaagtgtatcatatgccaa gtccgccccc tattgacgtc 420 aatgacggta aatggcccgc ctggcattatgcccagtaca tgaccttacg ggactttcct 480 acttggcagt acatctacgt attagtcatcgctattacca tggtgatgcg gttttggcag 540 tacaccaatg ggcgtggata gcggtttgactcacggggat ttccaagtct ccaccccatt 600 gacgtcaatg ggagtttgtt ttggcaccaaaatcaacggg actttccaaa atgtcgtaac 660 aactgcgatc gcccgccccg ttgacgcaaatgggcggtag gcgtgtacgg tgggaggtct 720 atataagcag agctcgttta gtgaaccgtcagatcactga attctgacga cctactgatt 780 aacggccaga tctaagctag cttcctgaaagatgaagcta ctgtcttcta tcgaacaagc 840 atgcgatatt tgccgactta aaaagctcaagtgctccaaa gaaaaaccga agtgcgccaa 900 gtgtctgaag aacaactggg agtgtcgctactctcccaaa accaaaaggt ctccgctgac 960 tagggcacat ctgacagaag tggaatcaaggctagaaaga ctggaacagc tatttctact 1020 gatttttcct cgagaagacc ttgacatgattttgaaaatg gattctttac aggatataaa 1080 agcattgtta acaggattat ttgtacaagataatgtgaat aaagatgccg tcacagatag 1140 attggcttca gtggagactg atatgcctctaacattgaga cagcatagaa taagtgcgac 1200 atcatcatcg gaagagagta gtaacaaaggtcaaagacag ttgactgtat cgccggaatt 1260 caggtgagta ctcgctacct taaggcctatctggccgttt aaacagatgt gtataagaga 1320 cagctctctt aaggtagcct gtctcttatacacatctaga tccttgctag agtcgaccaa 1380 ttctcatgtt tgacagctta tcatcgcagatcctgagctt gtatggtgca ctctcagtac 1440 aatctgctct gctgccgcat agttaagccagtatctgctc cctgcttgtg tgttggaggt 1500 cgctgagtag tgcgcgagca aaatttaagctacaacaagg caaggcttga ccgacaattg 1560 catgaagaat ctgcttaggg ttaggcgttttgcgctgctt cgcgatgtac gggccagata 1620 tacgcgtatc tgaggggact agggtgtgtttaggcgccca gcggggcttc ggttgtacgc 1680 ggttaggagt cccctcagga tatagtagtttcgcttttgc atagggaggg ggaaatgtag 1740 tcttatgcaa tacacttgta gtcttgcaacatggtaacga tgagttagca acatgcctta 1800 caaggagaga aaaagcaccg tgcatgccgattggtggaag taaggtggta cgatcgtgcc 1860 ttattaggaa ggcaacagac aggtctgacatggattggac gaaccactga attccgcatt 1920 gcagagataa ttgtatttaa gtgcctagctcgatacaata aacgccattt gaccattcac 1980 cacattggtg tgcacctcca agctgggtaccagctgctag cctcgagacg cgtgatttcc 2040 ttcgaagctt gtcatggttg gttcgctaaactgcatcgtc gctgtgtccc agaacatggg 2100 catcggcaag aacggggacc tgccctggccaccgctcagg aatgaattca gatatttcca 2160 gagaatgacc acaacctctt cagtagaaggtaaacagaat ctggtgatta tgggtaagaa 2220 gacctggttc tccattcctg agaagaatcgacctttaaag ggtagaatta atttagttct 2280 cagcagagaa ctcaaggaac ctccacaaggagctcatttt ctttccagaa gtctagatga 2340 tgccttaaaa cttactgaac aaccagaattagcaaataaa gtagacatgg tctggatagt 2400 tggtggcagt tctgtttata aggaagccatgaatcaccca ggccatctta aactatttgt 2460 gacaaggatc atgcaagact ttgaaagtgacacgtttttt ccagaaattg atttggagaa 2520 atataaactt ctgccagaat acccaggtgttctctctgat gtccaggagg agaaaggcat 2580 taagtacaaa tttgaagtat atgagaagaatgttaattaa gggcaccaat aactgcctta 2640 aaaaaattac gccccgccct gccactcatcgcagtactgt tgtaattcat taagcattct 2700 gccgacatgg aagccatcac agacggcatgatgaacctga atcgccagcg gcatcagcac 2760 cttgtcgcct tgcgtataat atttgcccatggtgaaaacg ggggcgaaga agttgtccat 2820 attggccacg tttaaatcaa aactggtgaaactcacccag ggattggctg agacgaaaaa 2880 catattctca ataaaccctt tagggaaataggccaggttt tcaccgtaac acgccacatc 2940 ttgcgaatat atgtgtagaa actgccggaaatcgtcgtgg tattcactcc agagcgatga 3000 aaacgtttca gtttgctcat ggaaaacggtgtaacaaggg tgaacactat cccatatcac 3060 cagctcaccg tctttcattg ccatacggaattccggatga gcattcatca ggcgggcaag 3120 aatgtgaata aaggccggat aaaacttgtgcttatttttc tttacggtct ttaaaaaggc 3180 cgtaatatcc agctgaacgg tctggttataggtacattga gcaactgact gaaatgcctc 3240 aaaatgttct ttacgatgcc attgggatatatcaacggtg gtatatccag tgattttttt 3300 ctccatttta gcttccttag ctcctgaaaatctcgataac tcaaaaaata cgcccggtag 3360 tgatcttatt tcattatggt gaaagttggaacctcttacg tgccgatcaa cgtctcattt 3420 tcgccaaatt aattaaggcg cgccgctctcctggctagga gtcacgtaga aaggactacc 3480 gacgaaggaa cttgggtcgc cggtgtgttcgtatatggag gtagtaagac ctccctttac 3540 aacctaaggc gaggaactgc ccttgctattccacaatgtc gtcttacacc attgagtcgt 3600 ctcccctttg gaatggcccc tggacccggcccacaacctg gcccgctaag ggagtccatt 3660 gtctgttatt tcatggtctt tttacaaactcatatatttg ctgaggtttt gaaggatgcg 3720 attaaggacc ttgttatgac aaagcccgctcctacctgca atatcagggt gactgtgtgc 3780 agctttgacg atggagtaga tttgcctccctggtttccac ctatggtgga aggggctgcc 3840 gcggagggtg atgacggaga tgacggagatgaaggaggtg atggagatga gggtgaggaa 3900 gggcaggagt gatgtaactt gttaggagacgccctcaatc gtattaaaag ccgtgtattc 3960 ccccgcacta aagaataaat ccccagtagacatcatgcgt gctgttggtg tatttctggc 4020 catctgtctt gtcaccattt tcgtcctcccaacatggggc aattgggcat acccatgttg 4080 tcacgtcact cagctccgcg ctcaacaccttctcgcgttg gaaaacatta gcgacattta 4140 cctggtgagc aatcagacat gcgacggctttagcctggcc tccttaaatt cacctaagaa 4200 tgggagcaac cagcatgcag gaaaaggacaagcagcgaaa attcacgccc ccttgggagg 4260 tggcggcata tgcaaaggat agcactcccactctactact gggtatcata tgctgactgt 4320 atatgcatga ggatagcata tgctacccggatacagatta ggatagcata tactacccag 4380 atatagatta ggatagcata tgctacccagatatagatta ggatagccta tgctacccag 4440 atataaatta ggatagcata tactacccagatatagatta ggatagcata tgctacccag 4500 atatagatta ggatagccta tgctacccagatatagatta ggatagcata tgctacccag 4560 atatagatta ggatagcata tgctatccagatatttgggt agtatatgct acccagatat 4620 aaattaggat agcatatact accctaatctctattaggat agcatatgct acccggatac 4680 agattaggat agcatatact acccagatatagattaggat agcatatgct acccagatat 4740 agattaggat agcctatgct acccagatataaattaggat agcatatact acccagatat 4800 agattaggat agcatatgct acccagatatagattaggat agcctatgct acccagatat 4860 agattaggat agcatatgct atccagatatttgggtagta tatgctaccc atggcaacat 4920 tagcccaccg tgctctcagc gacctcgtgaatatgaggac caacaaccct gtgcttggcg 4980 ctcaggcgca agtgtgtgta atttgtcctccagatcgcag caatcgcgcc cctatcttgg 5040 cccgcccacc tacttatgca ggtattccccggggtgccat tagtggtttt gtgggcaagt 5100 ggtttgaccg cagtggttag cggggttacaatcagccaag ttattacacc cttattttac 5160 agtccaaaac cgcagggcgg cgtgtgggggctgacgcgtg cccccactcc acaatttcaa 5220 aaaaaagagt ggccacttgt ctttgtttatgggccccatt ggcgtggagc cccgtttaat 5280 tttcgggggt gttagagaca accagtggagtccgctgctg tcggcgtcca ctctctttcc 5340 ccttgttaca aatagagtgt aacaacatggttcacctgtc ttggtccctg cctgggacac 5400 atcttaataa ccccagtatc atattgcactaggattatgt gttgcccata gccataaatt 5460 cgtgtgagat ggacatccag tctttacggcttgtccccac cccatggatt tctattgtta 5520 aagatattca gaatgtttca ttcctacactagtatttatt gcccaagggg tttgtgaggg 5580 ttatattggt gtcatagcac aatgccaccactgaaccccc cgtccaaatt ttattctggg 5640 ggcgtcacct gaaaccttgt tttcgagcacctcacataca ccttactgtt cacaactcag 5700 cagttattct attagctaaa cgaaggagaatgaagaagca ggcgaagatt caggagagtt 5760 cactgcccgc tccttgatct tcagccactgcccttgtgac taaaatggtt cactaccctc 5820 gtggaatcct gaccccatgt aaataaaaccgtgacagctc atggggtggg agatatcgct 5880 gttccttagg acccttttac taaccctaattcgatagcat atgcttcccg ttgggtaaca 5940 tatgctattg aattagggtt agtctggatagtatatacta ctacccggga agcatatgct 6000 acccgtttag ggttaacaag ggggccttataaacactatt gctaatgccc tcttgagggt 6060 ccgcttatcg gtagctacac aggcccctctgattgacgtt ggtgtagcct cccgtagtct 6120 tcctgggccc ctgggaggta catgtcccccagcattggtg taagagcttc agccaagagt 6180 tacacataaa ggcaatgttg tgttgcagtccacagactgc aaagtctgct ccaggatgaa 6240 agccactcag tgttggcaaa tgtgcacatccatttataag gatgtcaact acagtcagag 6300 aacccctttg tgtttggtcc ccccccgtgtcacatgtgga acagggccca gttggcaagt 6360 tgtaccaacc aactgaaggg attacatgcactgccccgaa tacaaaacaa aagcgctcct 6420 cgtaccagcg aagaaggggc agagatgccgtagtcaggtt tagttcgtcc ggcggcgggc 6480 ggccgcaagg cgcgccggat ccacaggacgggtgtggtcg ccatgatcgc gtagtcgata 6540 gtggctccaa gtagcgaagc gagcaggactgggcggcggc caaagcggtc ggacagtgct 6600 ccgagaacgg gtgcgcatag aaattgcatcaacgcatata gcgctagatc cttgctagag 6660 tcgagatctg tcgagccatg tgagcaaaaggccagcaaaa ggccaggaac cgtaaaaagg 6720 ccgcgttgct ggcgtttttc cataggctccgcccccctga cgagcatcac aaaaatcgac 6780 gctcaagtca gaggtggcga aacccgacaggactataaag ataccaggcg tttccccctg 6840 gaagctccct cgtgcgctct cctgttccgaccctgccgct taccggatac ctgtccgcct 6900 ttctcccttc gggaagcgtg gcgctttctcatagctcacg ctgtaggtat ctcagttcgg 6960 tgtaggtcgt tcgctccaag ctgggctgtgtgcacgaacc ccccgttcag cccgaccgct 7020 gcgccttatc cggtaactat cgtcttgagtccaacccggt aagacacgac ttatcgccac 7080 tggcagcagc cactggtaac aggattagcagagcgaggta tgtaggcggt gctacagagt 7140 tcttgaagtg gtggcctaac tacggctacactagaaggac agtatttggt atctgcgctc 7200 tgctgaagcc agttaccttc ggaaaaagagttggtagctc ttgatccggc aaacaaacca 7260 ccgctggtag cggtggtttt tttgtttgcaagcagcagat tacgcgcaga aaaaaaggat 7320 ctcaagaaga tcctttgatc ttttctacggggtctgacgc tcagtggaac gaaaactcac 7380 gttaagggat tttggtcatg agattatcaaaaaggatctt cacctagatc cttttatcgg 7440 tgtgaaatac cgcacagatg cgtaaggagaaaataccgca tcaggaaatt gtaagcgtta 7500 ataattcaga agaactcgtc aagaaggcgatagaaggcga tgcgctgcga atcgggagcg 7560 gcgataccgt aaagcacgag gaagcggtcagcccattcgc cgccaagctc ttcagcaata 7620 tcacgggtag ccaacgctat gtcctgatagcggtccgcca cacccagccg gccacagtcg 7680 atgaatccag aaaagcggcc attttccaccatgatattcg gcaagcaggc atcgccatgg 7740 gtcacgacga gatcctcgcc gtcgggcatgctcgccttga gcctggcgaa cagttcggct 7800 ggcgcgagcc cctgatgctc ttcgtccagatcatcctgat cgacaagacc ggcttccatc 7860 cgagtacgtg ctcgctcgat gcgatgtttcgcttggtggt cgaatgggca ggtagccgga 7920 tcaagcgtat gcagccgccg cattgcatcagccatgatgg atactttctc ggcaggagca 7980 aggtgagatg acaggagatc ctgccccggcacttcgccca atagcagcca gtcccttccc 8040 gcttcagtga caacgtcgag cacagctgcgcaaggaacgc ccgtcgtggc cagccacgat 8100 agccgcgctg cctcgtcttg cagttcattcagggcaccgg acaggtcggt cttgacaaaa 8160 agaaccgggc gcccctgcgc tgacagccggaacacggcgg catcagagca gccgattgtc 8220 tgttgtgccc agtcatagcc gaatagcctctccacccaag cggccggaga acctgcgtgc 8280 aatccatctt gttcaatcat gcgaaacgatcctcatcctg tctcttgatc agagcttgat 8340 cccctgcgcc atcagatcct tggcggcgagaaagccatcc agtttacttt gcagggcttg 8400 tcaaccttac cagataaaag tgctcatcattggaaaacat tcaattcgtc gacctcgaaa 8460 ttctaccggg taggggaggc gcttttcccaaggcagtctg gagcatgcgc tttagcagcc 8520 ccgctgggca cttggcgcta cacaagtggcctctggcctc gcacacattc cacatccacc 8580 ggtaggcgcc aaccggctcc gttctttggtggccccttcg cgccaccttc tactcctccc 8640 ctagtcagga agttcccccc cgccccgcanctcgcgtcgt gcaggacgtg acaaatggaa 8700 atagcacgtc tcactagtct cgtgcagatggacaagcacc gctgagcaat ggagcgggta 8760 ggcctttggg gcagcggcca atagcagctttgctccttcg ctttctgggc tcagaggctg 8820 gnaaggggtg ggtccggggg cgggctcaggggcgggctca ggggcggggc gggcgcccga 8880 aggtcctccg gaggcccggc attctgcacgcttcaaaagc gcacgtctgc cgcgctgttc 8940 tcctcttcct catctccggg cctttcgacctgcatccatc tagatctcga gcagctgaag 9000 cttaccatga ccgagtacaa gcccacggtgcgcctcgcca cccgcgacga cgtcccccgg 9060 gccgtacgca ccctcgccgc cgcgttcgccgactaccccg ccacgcgcca caccgtcgac 9120 ccggaccgcc acatcgagcg ggtcaccgagctgcaagaac tcttcctcac gcgcgtcggg 9180 ctcgacatcg gcaaggtgtg ggtcgcggacgacggcgccg cggtggcggt ctggaccacg 9240 ccggagagcg tcgaagcggg ggcggtgttcgccgagatcg gcccgcgcat ggccgagttg 9300 agcggttccc ggctggccgc gcagcaacagatggaaggcc tcctggcgcc gcaccgggcc 9360 caaggagccc gcgtggttcc ttggcccaccgtcgggcgtc ttcgcccgac caccagggca 9420 agggtctggc aagcgccgtc gtgctccccggagtggaggc ggccgagcgc gccggggtgc 9480 ccgccttcct ggagacctcc gcgccccgcaacctcccctt ctacgagcgg ctcggcttca 9540 ccgtcaccgc cgacgtcgag gtgcccgaaggaccgcgcac ctggtgcatg acccgcaagc 9600 ccggtgcctg acgcccgccc cacgacccgcagcgcccgac cgaaaggagc gcacgacccc 9660 atgcatcgat ggcactgggc aggtaagtatcaaggttagc ggccgctaac ctggttgctg 9720 actaattgag atgcatgctt tgcatacttctgcctgctgg ggagcctggg gactttccac 9780 accctaactg acacacattc cacagctggttctttccgcc tcagaaggta cacaggcgaa 9840 attgtaagcg ttaatatttt gttaaaattcgcgttaaatt tttgttaaat cagctcattt 9900 tttaaccaat aggccgaaat cggcaaaatcccttataaat caaaagaata gaccgagata 9960 gggttgagtg ttgttccagt ttggaacaagagtccactat taaagaacgt ggactccaac 10020 gtcaaagggc gaaaaaccgt ctatcagggcgatggcccac 10060 26 7714 DNA Homo sapiens 26 tcaacgacag gagcacgatcatgcgcaccc gtggccagga cccaacgctg cccgagatgc 60 gccgcgtgcg gctgctggagatggcggacg cgatggatat gttctgccaa gggttggttt 120 gcgcattcac agttctccgcaagaattgat tggctccaat tcttggagtg gtgaatccgt 180 tagcgaggtg ccgccggcttccattcaggt cgaggtggcc cggctccatg caccgcgacg 240 caacgcgggg aggcagacaaggtatagggc ggcgcctaca atccatgcca acccgttcca 300 tgtgctcgcc gaggcggcataaatcgccgt gacgatcagc ggtccagtga tcgaagttag 360 gctggtaaga gccgcgagcgatccttgaag ctgtccctga tggtcgtcat ctacctgcct 420 ggacagcatg gcctgcaacgcgggcatccc gatgccgccg gaagcgagaa gaatcataat 480 ggggaaggcc atccagcctcgcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc 540 cgccatgccg gcgataatggcctgcttctc gccgaaacgt ttggtggcgg gaccagtgac 600 gaaggcttga gcgagggcgtgcaagattcc gaataccgca agcgacaggc cgatcatcgt 660 cgcgctccag cgaaagcggtcctcgccgaa aatgacccag agcgctgccg gcacctgtcc 720 tacgagttgc atgataaagaagacagtcat aagtgcggcg acgatagtca tgccccgcgc 780 ccaccggaag gagctgactgggttgaaggc tctcaagggc atcggtcgac gctctccctt 840 atgcgactcc tgcattaggaagcagcccag tagtaggttg aggccgttga gcaccgccgc 900 cgcaaggaat ggtgcatgcaaggagatggc gcccaacagt cccccggcca cggggcctgc 960 caccataccc acgccgaaacaagcgctcat gagcccgaag tggcgagccc gatcttcccc 1020 atcggtgatg tcggcgatataggcgccagc aaccgcacct gtggcgccgg tgatgccggc 1080 cacgatgcgt ccggcgtagaggatccacag gacgggtgtg gtcgccatga tcgcgtagtc 1140 gatagtggct ccaagtagcgaagcgagcag gactgggcgg cggccaaagc ggtcggacag 1200 tgctccgaga acgggtgcgcatagaaattg catcaacgca tatagcgcta gcagcacgcc 1260 atagtgactg gcgatgctgtcggaatggac gatatcccgc aagaggcccg gcagtaccgg 1320 cataaccaag cctatgcctacagcatccag ggtgacggtg ccgaggatga cgatgagcgc 1380 attgttagat ttcatacacggtgcctgact gcgttagcaa tttaactgtg ataaactacc 1440 gcattaaagc ttatcgatttccacacatta tacgagccga tgttaattgt caacagctca 1500 tgcatgacgt cccgggagcagacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg 1560 tcggggctgg cttaactatgcggcatcaga gcagattgta ctgagagtgc accatatgcg 1620 gtgtgaaata ccgcacagatgcgtaaggag aaaataccgc atcaggcgcc attcgccatt 1680 caggctgcgc aactgttgggaagggcgatc ggtgcgggcc tcttcgctat tacgccagct 1740 ggcgaaaggg ggatgtgctgcaaggcgatt aagttgggta acgccagggt tttcccagtc 1800 acgacgttgt aaaacgacggccagtgaatt cgagctcata cttcgaatag ggataacagg 1860 gtaatgcgat agcggccgcaatcgctctct taaggtagcc cgtgctggca aacagctatt 1920 atgggtatta tgggtgggccctagaaagct tggcgtaatc atggtcatag ctgtttcctg 1980 tgtgaaattg ttatccgctcacaattccac acaacatacg agccggaagc ataaagtgta 2040 aagcctgggg tgcctaatgagtgagctaac tcacattaat tgcgttgcgc tcactgcccg 2100 ctttccagtc gggaaacctgtcgtgccagc tgcattaatg acccgcgagg tcgccgcccc 2160 gtaaccccct accgctgaaagttctgcaaa gcctgatggg acataagtcc atcagttcaa 2220 cggaagtcta cacgaaggtttttgcgctgg atgtggctgc ccggcaccgg gtgcagtttg 2280 cgatgccgga gtctgatgcggttgcgatgc tgaaacaatt atcctgagaa taaatgcctt 2340 ggcctttata tggaaatgtggaactgagtg gatatgctgt ttttgtctgt taaacagaga 2400 agctggctgt tatccactgagaagcgaacg aaacagtcgg gaaaatctcc cattatcgta 2460 gagatccgca ttattaatctcaggagcctg tgtagcgttt ataggaagta gtgttctgtc 2520 atgatgcctg caagcggtaacgaaaacgat ttgaatatgc cttcaggaac aatagaaatc 2580 ttcgtgcggt gttacgttgaagtggagcgg attatgtcag caatggacag aacaacctaa 2640 tgaacacaga accatgatgtggtctgtcct tttacagcca gtagtgctcg ccgcagtcga 2700 gcgacagggc gaagccctcgagtgagcgag gaagcaccag ggaacagcac ttatatattc 2760 tgcttacaca cgatgcctgaaaaaacttcc cttggggtta tccacttatc cacggggata 2820 tttttataat tattttttttatagttttta gatcttcttt tttagagcgc cttgtaggcc 2880 tttatccatg ctggttctagagaaggtgtt gtgacaaatt gccctttcag tgtgacaaat 2940 caccctcaaa tgacagtcctgtctgtgaca aattgccctt aaccctgtga caaattgccc 3000 tcagaagaag ctgttttttcacaaagttat ccctgcttat tgactctttt ttatttagtg 3060 tgacaatcta aaaacttgtcacacttcaca tggatctgtc atggcggaaa cagcggttat 3120 caatcacaag aaacgtaaaaatagcccgcg aatcgtccag tcaaacgacc tcactgaggc 3180 ggcatatagt ctctcccgggatcaaaaacg tatgctgtat ctgttcgttg accagatcag 3240 aaaatctgat ggcaccctacaggaacatga cggtatctgc gagatccatg ttgctaaata 3300 tgctgaaata ttcggattgacctctgcgga agccagtaag gatatacggc aggcattgaa 3360 gagtttcgcg gggaaggaagtggtttttta tcgccctgaa gaggatgccg gcgatgaaaa 3420 aggctatgaa tcttttccttggtttatcaa acgtgcgcac agtccatcca gagggcttta 3480 cagtgtacat atcaacccatatctcattcc cttctttatc gggttacaga accggtttac 3540 gcagtttcgg cttagtgaaacaaaagaaat caccaatccg tatgccatgc gtttatacga 3600 atccctgtgt cagtatcgtaagccggatgg ctcaggcatc gtctctctga aaatcgactg 3660 gatcatagag cgttaccagctgcctcaaag ttaccagcgt atgcctgact tccgccgccg 3720 cttcctgcag gtctgtgttaatgagatcaa cagcagaact ccaatgcgcc tctcatacat 3780 tgagaaaaag aaaggccgccagacgactca tatcgtattt tccttccgcg atatcacttc 3840 catgacgaca ggatagtctgagggttatct gtcacagatt tgagggtggt tcgtcacatt 3900 tgttctgacc tactgagggtaatttgtcac agttttgctg tttccttcag cctgcatgga 3960 ttttctcata ctttttgaactgtaattttt aaggaagcca aatttgaggg cagtttgtca 4020 cagttgattt ccttctctttcccttcgtca tgtgacctga tatcgggggt tagttcgtca 4080 tcattgatga gggttgattatcacagttta ttactctgaa ttggctatcc gcgtgtgtac 4140 ctctacctgg agtttttcccacggtggata tttcttcttg cgctgagcgt aagagctatc 4200 tgacagaaca gttcttctttgcttcctcgc cagttcgctc gctatgctcg gttacacggc 4260 tgcggcgagc gctagtgataataagtgact gaggtatgtg ctcttcttat ctccttttgt 4320 agtgttgctc ttattttaaacaactttgcg gttttttgat gactttgcga ttttgttgtt 4380 gctttgcagt aaattgcaagatttaataaa aaaacgcaaa gcaatgatta aaggatgttc 4440 agaatgaaac tcatggaaacacttaaccag tgcataaacg ctggtcatga aatgacgaag 4500 gctatcgcca ttgcacagtttaatgatgac agcccggaag cgaggaaaat aacccggcgc 4560 tggagaatag gtgaagcagcggatttagtt ggggtttctt ctcaggctat cagagatgcc 4620 gagaaagcag ggcgactaccgcacccggat atggaaattc gaggacgggt tgagcaacgt 4680 gttggttata caattgaacaaattaatcat atgcgtgatg tgtttggtac gcgattgcga 4740 cgtgctgaag acgtatttccaccggtgatc ggggttgctg cccataaagg tggcgtttac 4800 aaaacctcag tttctgttcatcttgctcag gatctggctc tgaaggggct acgtgttttg 4860 ctcgtggaag gtaacgacccccagggaaca gcctcaatgt atcacggatg ggtaccagat 4920 cttcatattc atgcagaagacactctcctg cctttctatc ttggggaaaa ggacgatgtc 4980 acttatgcaa taaagcccacttgctggccg gggcttgaca ttattccttc ctgtctggct 5040 ctgcaccgta ttgaaactgagttaatgggc aaatttgatg aaggtaaact gcccaccgat 5100 ccacacctga tgctccgactggccattgaa actgttgctc atgactatga tgtcatagtt 5160 attgacagcg cgcctaacctgggtatcggc acgattaatg tcgtatgtgc tgctgatgtg 5220 ctgattgttc ccacgcctgctgagttgttt gactacacct ccgcactgca gtttttcgat 5280 atgcttcgtg atctgctcaagaacgttgat cttaaagggt tcgagcctga tgtacgtatt 5340 ttgcttacca aatacagcaatagtaatggc tctcagtccc cgtggatgga ggagcaaatt 5400 cgggatgcct ggggaagcatggttctaaaa aatgttgtac gtgaaacgga tgaagttggt 5460 aaaggtcaga tccggatgagaactgttttt gaacaggcca ttgatcaacg ctcttcaact 5520 ggtgcctgga gaaatgctctttctatttgg gaacctgtct gcaatgaaat tttcgatcgt 5580 ctgattaaac cacgctgggagattagataa tgaagcgtgc gcctgttatt ccaaaacata 5640 cgctcaatac tcaaccggttgaagatactt cgttatcgac accagctgcc ccgatggtgg 5700 attcgttaat tgcgcgcgtaggagtaatgg ctcgcggtaa tgccattact ttgcctgtat 5760 gtggtcggga tgtgaagtttactcttgaag tgctccgggg tgatagtgtt gagaagacct 5820 ctcgggtatg gtcaggtaatgaacgtgacc aggagctgct tactgaggac gcactggatg 5880 atctcatccc ttcttttctactgactggtc aacagacacc ggcgttcggt cgaagagtat 5940 ctggtgtcat agaaattgccgatgggagtc gccgtcgtaa agctgctgca cttaccgaaa 6000 gtgattatcg tgttctggttggcgagctgg atgatgagca gatggctgca ttatccagat 6060 tgggtaacga ttatcgcccaacaagtgctt atgaacgtgg tcagcgttat gcaagccgat 6120 tgcagaatga atttgctggaaatatttctg cgctggctga tgcggaaaat atttcacgta 6180 agattattac ccgctgtatcaacaccgcca aattgcctaa atcagttgtt gctctttttt 6240 ctcaccccgg tgaactatctgcccggtcag gtgatgcact tcaaaaagcc tttacagata 6300 aagaggaatt acttaagcagcaggcatcta accttcatga gcagaaaaaa gctggggtga 6360 tatttgaagc tgaagaagttatcactcttt taacttctgt gcttaaaacg tcatctgcat 6420 caagaactag tttaagctcacgacatcagt ttgctcctgg agcgacagta ttgtataagg 6480 gcgataaaat ggtgcttaacctggacaggt ctcgtgttcc aactgagtgt atagagaaaa 6540 ttgaggccat tcttaaggaacttgaaaagc cagcaccctg atgcgaccac gttttagtct 6600 acgtttatct gtctttacttaatgtccttt gttacaggcc agaaagcata actggcctga 6660 atattctctc tgggccagaagcttggccca ctgttccact tgtatcgtcg gtctgataat 6720 cagactggga ccacggtcccactcgtatcg tcggtctgat tattagtctg ggaccacggt 6780 cccactcgta tcgtcggtctgattattagt ctgggaccac ggtcccactc gtatcgtcgg 6840 tctgataatc agactgggaccacggtccca ctcgtatcgt cggtctgatt attagtctgg 6900 gaccatggtc ccactcgtatcgtcggtctg attattagtc tgggaccacg gtcccactcg 6960 tatcgtcggt ctgattattagtctggaacc acggtcccac tcgtatcgtc ggtctgatta 7020 ttagtctggg accacggtcccactcgtatc gtcggtctga ttattagtct gggaccacga 7080 tcccactcgt gttgtcggtctgattatcgg tctgggacca cggtcccact tgtattgtcg 7140 atcagactat cagcgtgagactacgattcc atcaatgcct gtcaagggca agtattgaca 7200 tgtcgtcgta acctgtagaacggagtaacc tcggtgtgcg gttgtatgcc tgctgtggat 7260 tgctgctgtg tcctgcttatccacaacatt ttgcgcacgg ttatgtggac aaaatacctg 7320 cgctagagaa aagagtttgtagaaacgcaa aaaggccatc cgtcaggatg gccttctgct 7380 taatttgatg cctggcagtttatggcgggc gtcctgcccg ccaccctccg ggccgttgct 7440 tcgcaacgtt caaatccgctcccggcggat ttgtcctact caggagagcg ttcaccgaca 7500 aacaacagat aaaacgaaaggcccagtctt tcgactgagc ctttcgtttt atttgatgcc 7560 tggcagttcc ctactctcgcatggggagac cccacactac catcggcgct acggcgtttc 7620 acttctgagt tcggcatggggtcaggtggg accaccgcgc tactgccgcc aggcaaattc 7680 tgttttatca gaccgcttctgcgttctggg ccgc 7714 27 5314 DNA Homo sapiens 27 gatcttcaat attggccattagccatatta ttcattggtt atatagcata aatcaatatt 60 ggctattggc cattgcatacgttgtatcta tatcataata tgtacattta tattggctca 120 tgtccaatat gaccgccatgttggcattga ttattgacta gttattaata gtaatcaatt 180 acggggtcat tagttcatagcccatatatg gagttccgcg ttacataact tacggtaaat 240 ggcccgcctg gctgaccgcccaacgacccc cgcccattga cgtcaataat gacgtatgtt 300 cccatagtaa cgccaatagggactttccat tgacgtcaat gggtggagta tttacggtaa 360 actgcccact tggcagtacatcaagtgtat catatgccaa gtccgccccc tattgacgtc 420 aatgacggta aatggcccgcctggcattat gcccagtaca tgaccttacg ggactttcct 480 acttggcagt acatctacgtattagtcatc gctattacca tggtgatgcg gttttggcag 540 tacaccaatg ggcgtggatagcggtttgac tcacggggat ttccaagtct ccaccccatt 600 gacgtcaatg ggagtttgttttggcaccaa aatcaacggg actttccaaa atgtcgtaac 660 aactgcgatc gcccgccccgttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 720 atataagcag agctcgtttagtgaaccgtc agatcactga attctgacga cctactgatt 780 aacggccata gaggcctcctgcagaactgt cttagtgaca actatcgatt tccacacatt 840 atacgagccg atgttaattgtcaacagctc atgcatgacg tcccgggagc agacaagccc 900 gaccatggct cgagtaatacgactcactat agggcgacag gtgagtactc gctaccttaa 960 gagaggccta tctggccagttagcagtcga agaaagaagt ttaagagagc cgaaacaagc 1020 gctcatgagc ccgaagtggcgagcccgatc ttccccatcg gtgatgtcgg cgatataggc 1080 gccagcaacc gcacctgtggcgccggtgat gccggccacg atgcgtccgg cgtagaggat 1140 ccacaggacg ggtgtggtcgccatgatcgc gtagtcgata gtggctccaa gtagcgaagc 1200 gagcaggact gggcggcggccaaagcggtc ggacagtgct ccgagaacgg gtgcgcatag 1260 aaattgcatc aacgcatatagcgctagatc cttgctagag tcgagatctg tcgagccatg 1320 tgagcaaaag gccagcaaaaggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 1380 cataggctcc gcccccctgacgagcatcac aaaaatcgac gctcaagtca gaggtggcga 1440 aacccgacag gactataaagataccaggcg tttccccctg gaagctccct cgtgcgctct 1500 cctgttccga ccctgccgcttaccggatac ctgtccgcct ttctcccttc gggaagcgtg 1560 gcgctttctc atagctcacgctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 1620 ctgggctgtg tgcacgaaccccccgttcag cccgaccgct gcgccttatc cggtaactat 1680 cgtcttgagt ccaacccggtaagacacgac ttatcgccac tggcagcagc cactggtaac 1740 aggattagca gagcgaggtatgtaggcggt gctacagagt tcttgaagtg gtggcctaac 1800 tacggctaca ctagaaggacagtatttggt atctgcgctc tgctgaagcc agttaccttc 1860 ggaaaaagag ttggtagctcttgatccggc aaacaaacca ccgctggtag cggtggtttt 1920 tttgtttgca agcagcagattacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 1980 ttttctacgg ggtctgacgctcagtggaac gaaaactcac gttaagggat tttggtcatg 2040 agattatcaa aaaggatcttcacctagatc cttttatcgg tgtgaaatac cgcacagatg 2100 cgtaaggaga aaataccgcatcaggaaatt gtaagcgtta ataattcaga agaactcgtc 2160 aagaaggcga tagaaggcgatgcgctgcga atcgggagcg gcgataccgt aaagcacgag 2220 gaagcggtca gcccattcgccgccaagctc ttcagcaata tcacgggtag ccaacgctat 2280 gtcctgatag cggtccgccacacccagccg gccacagtcg atgaatccag aaaagcggcc 2340 attttccacc atgatattcggcaagcaggc atcgccatgg gtcacgacga gatcctcgcc 2400 gtcgggcatg ctcgccttgagcctggcgaa cagttcggct ggcgcgagcc cctgatgctc 2460 ttcgtccaga tcatcctgatcgacaagacc ggcttccatc cgagtacgtg ctcgctcgat 2520 gcgatgtttc gcttggtggtcgaatgggca ggtagccgga tcaagcgtat gcagccgccg 2580 cattgcatca gccatgatggatactttctc ggcaggagca aggtgagatg acaggagatc 2640 ctgccccggc acttcgcccaatagcagcca gtcccttccc gcttcagtga caacgtcgag 2700 cacagctgcg caaggaacgcccgtcgtggc cagccacgat agccgcgctg cctcgtcttg 2760 cagttcattc agggcaccggacaggtcggt cttgacaaaa agaaccgggc gcccctgcgc 2820 tgacagccgg aacacggcggcatcagagca gccgattgtc tgttgtgccc agtcatagcc 2880 gaatagcctc tccacccaagcggccggaga acctgcgtgc aatccatctt gttcaatcat 2940 gcgaaacgat cctcatcctgtctcttgatc agagcttgat cccctgcgcc atcagatcct 3000 tggcggcgag aaagccatccagtttacttt gcagggcttg tcaaccttac cagataaaag 3060 tgctcatcat tggaaaacgttcaattctga ggcggaaaga accagctgtg gaatgtgtgt 3120 cagttagggt gtggaaagtccccaggctcc ccagcaggca gaagtatgca aagcatgcat 3180 ctcaattagt cagcaaccaggtgtggaaag tccccaggct ccccagcagg cagaagtatg 3240 caaagcatgc atctcaattagtcagcaacc atagtcccgc ccctaactcc gcccatcccg 3300 cccctaactc cgcccagttccgcccattct ccgccccatg gctgactaat tttttttatt 3360 tatgcagagg ccgaggccgcctcggcctct gagctattcc agaagtagtg aggaggcttt 3420 tttggaggcc taggcttttgcaaaaagctt gattcttctg acacaacagt ctcgaactta 3480 aggctagagc caccatgattgaacaagatg gattgcacgc aggttctccg gccgcttggg 3540 tggagaggct attcggctatgactgggcac aacagacaat cggctgctct gatgccgccg 3600 tgttccggct gtcagcgcaggggcgcccgg ttctttttgt caagaccgac ctgtccggtg 3660 ccctgaatga actgcaggacgaggcagcgc ggctatcgtg gctggccacg acgggcgttc 3720 cttgcgcagc tgtgctcgacgttgtcactg aagcgggaag ggactggctg ctattgggcg 3780 aagtgccggg gcaggatctcctgtcatctc accttgctcc tgccgagaaa gtatccatca 3840 tggctgatgc aatgcggcggctgcatacgc ttgatccggc tacctgccca ttcgaccacc 3900 aagcgaaaca tcgcatcgagcgagcacgta ctcggatgga agccggtctt gtcgatcagg 3960 atgatctgga cgaagagcatcaggggctcg cgccagccga actgttcgcc aggctcaagg 4020 cgcgcatgcc cgacggcgaggatctcgtcg tgacccatgg cgatgcctgc ttgccgaata 4080 tcatggtgga aaatggccgcttttctggat tcatcgactg tggccggctg ggtgtggcgg 4140 accgctatca ggacatagcgttggctaccc gtgatattgc tgaagagctt ggcggcgaat 4200 gggctgaccg cttcctcgtgctttacggta tcgccgctcc cgattcgcag cgcatcgcct 4260 tctatcgcct tcttgacgagccattctgct ggatggctac aggtcgcagc cctggcgtcg 4320 tgattagtga tgatgaaccaggttatgacc ttgatttatt ttgcatacct aatcattatg 4380 ctgaggattt ggaaagggtgtttattcctc atggactaat tatggacagg actgaacgtc 4440 ttgctcgaga tgtgatgaaggagatgggag gccatcacat tgtagccctc tgtgtgctca 4500 aggggggcta taaattctttgctgacctgc tggattacat caaagcactg aatagaaata 4560 gtgatagatc cattcctatgactgtagatt ttatcagact gaagagctat tgtaatgacc 4620 agtcaacagg ggacataaaagtaattggtg gagatgatct ctcaacttta actggaaaga 4680 atgtcttgat tgtggaagatataattgaca ctggcaaaac aatgcagact ttgctttcct 4740 tggtcaggca gtataatccaaagatggtca aggtcgcaag cttgctggtg aaaaggaccc 4800 cacgaagtgt tggatataagccagactttg ttggatttga aattccagac aagtttgttg 4860 taggatatgc ccttgactataatgaatact tcagggattt gaatcatgtt tgtgtcatta 4920 gtgaaactgg aaaagcaaaatacaaagcct aagcggccgc taacctggtt gctgactaat 4980 tgagatgcat gctttgcatacttctgcctg ctggggagcc tggggacttt ccacacccta 5040 actgacacac attccacagctggttctttc cgcctcagaa ggtacacagg cgaaattgta 5100 agcgttaata ttttgttaaaattcgcgtta aatttttgtt aaatcagctc attttttaac 5160 caataggccg aaatcggcaaaatcccttat aaatcaaaag aatagaccga gatagggttg 5220 agtgttgttc cagtttggaacaagagtcca ctattaaaga acgtggactc caacgtcaaa 5280 gggcgaaaaa ccgtctatcagggcgatggc ccac 5314 28 9737 DNA Homo sapiens modified_base (8347) a, c,t, g, other or unknown 28 gatcttcaat attggccatt agccatatta ttcattggttatatagcata aatcaatatt 60 ggctattggc cattgcatac gttgtatcta tatcataatatgtacattta tattggctca 120 tgtccaatat gaccgccatg ttggcattga ttattgactagttattaata gtaatcaatt 180 acggggtcat tagttcatag cccatatatg gagttccgcgttacataact tacggtaaat 240 ggcccgcctg gctgaccgcc caacgacccc cgcccattgacgtcaataat gacgtatgtt 300 cccatagtaa cgccaatagg gactttccat tgacgtcaatgggtggagta tttacggtaa 360 actgcccact tggcagtaca tcaagtgtat catatgccaagtccgccccc tattgacgtc 420 aatgacggta aatggcccgc ctggcattat gcccagtacatgaccttacg ggactttcct 480 acttggcagt acatctacgt attagtcatc gctattaccatggtgatgcg gttttggcag 540 tacaccaatg ggcgtggata gcggtttgac tcacggggatttccaagtct ccaccccatt 600 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacgggactttccaaa atgtcgtaac 660 aactgcgatc gcccgccccg ttgacgcaaa tgggcggtaggcgtgtacgg tgggaggtct 720 atataagcag agctcgttta gtgaaccgtc agatcactgaattctgacga cctactgatt 780 aacggccata gaggcctcct gcagaactgt cttagtgacaactatcgatt tccacacatt 840 atacgagccg atgttaattg tcaacagctc atgcatgacgtcccgggagc agacaagccc 900 gaccatggct cgagtaatac gactcactat agggcgacaggtgagtactc gctaccttaa 960 ggcctatctg gccgtttaaa cagatgtgta taagagacagctctcttaag gtagcctgtc 1020 tcttatacac atctagatcc ttgctagagt cgaccaattctcatgtttga cagcttatca 1080 tcgcagatcc tgagcttgta tggtgcactc tcagtacaatctgctctgct gccgcatagt 1140 taagccagta tctgctccct gcttgtgtgt tggaggtcgctgagtagtgc gcgagcaaaa 1200 tttaagctac aacaaggcaa ggcttgaccg acaattgcatgaagaatctg cttagggtta 1260 ggcgttttgc gctgcttcgc gatgtacggg ccagatatacgcgtatctga ggggactagg 1320 gtgtgtttag gcgcccagcg gggcttcggt tgtacgcggttaggagtccc ctcaggatat 1380 agtagtttcg cttttgcata gggaggggga aatgtagtcttatgcaatac acttgtagtc 1440 ttgcaacatg gtaacgatga gttagcaaca tgccttacaaggagagaaaa agcaccgtgc 1500 atgccgattg gtggaagtaa ggtggtacga tcgtgccttattaggaaggc aacagacagg 1560 tctgacatgg attggacgaa ccactgaatt ccgcattgcagagataattg tatttaagtg 1620 cctagctcga tacaataaac gccatttgac cattcaccacattggtgtgc acctccaagc 1680 tgggtaccag ctgctagcct cgagacgcgt gatttccttcgaagcttgtc atggttggtt 1740 cgctaaactg catcgtcgct gtgtcccaga acatgggcatcggcaagaac ggggacctgc 1800 cctggccacc gctcaggaat gaattcagat atttccagagaatgaccaca acctcttcag 1860 tagaaggtaa acagaatctg gtgattatgg gtaagaagacctggttctcc attcctgaga 1920 agaatcgacc tttaaagggt agaattaatt tagttctcagcagagaactc aaggaacctc 1980 cacaaggagc tcattttctt tccagaagtc tagatgatgccttaaaactt actgaacaac 2040 cagaattagc aaataaagta gacatggtct ggatagttggtggcagttct gtttataagg 2100 aagccatgaa tcacccaggc catcttaaac tatttgtgacaaggatcatg caagactttg 2160 aaagtgacac gttttttcca gaaattgatt tggagaaatataaacttctg ccagaatacc 2220 caggtgttct ctctgatgtc caggaggaga aaggcattaagtacaaattt gaagtatatg 2280 agaagaatgt taattaaggg caccaataac tgccttaaaaaaattacgcc ccgccctgcc 2340 actcatcgca gtactgttgt aattcattaa gcattctgccgacatggaag ccatcacaga 2400 cggcatgatg aacctgaatc gccagcggca tcagcaccttgtcgccttgc gtataatatt 2460 tgcccatggt gaaaacgggg gcgaagaagt tgtccatattggccacgttt aaatcaaaac 2520 tggtgaaact cacccaggga ttggctgaga cgaaaaacatattctcaata aaccctttag 2580 ggaaataggc caggttttca ccgtaacacg ccacatcttgcgaatatatg tgtagaaact 2640 gccggaaatc gtcgtggtat tcactccaga gcgatgaaaacgtttcagtt tgctcatgga 2700 aaacggtgta acaagggtga acactatccc atatcaccagctcaccgtct ttcattgcca 2760 tacggaattc cggatgagca ttcatcaggc gggcaagaatgtgaataaag gccggataaa 2820 acttgtgctt atttttcttt acggtcttta aaaaggccgtaatatccagc tgaacggtct 2880 ggttataggt acattgagca actgactgaa atgcctcaaaatgttcttta cgatgccatt 2940 gggatatatc aacggtggta tatccagtga tttttttctccattttagct tccttagctc 3000 ctgaaaatct cgataactca aaaaatacgc ccggtagtgatcttatttca ttatggtgaa 3060 agttggaacc tcttacgtgc cgatcaacgt ctcattttcgccaaattaat taaggcgcgc 3120 cgctctcctg gctaggagtc acgtagaaag gactaccgacgaaggaactt gggtcgccgg 3180 tgtgttcgta tatggaggta gtaagacctc cctttacaacctaaggcgag gaactgccct 3240 tgctattcca caatgtcgtc ttacaccatt gagtcgtctcccctttggaa tggcccctgg 3300 acccggccca caacctggcc cgctaaggga gtccattgtctgttatttca tggtcttttt 3360 acaaactcat atatttgctg aggttttgaa ggatgcgattaaggaccttg ttatgacaaa 3420 gcccgctcct acctgcaata tcagggtgac tgtgtgcagctttgacgatg gagtagattt 3480 gcctccctgg tttccaccta tggtggaagg ggctgccgcggagggtgatg acggagatga 3540 cggagatgaa ggaggtgatg gagatgaggg tgaggaagggcaggagtgat gtaacttgtt 3600 aggagacgcc ctcaatcgta ttaaaagccg tgtattcccccgcactaaag aataaatccc 3660 cagtagacat catgcgtgct gttggtgtat ttctggccatctgtcttgtc accattttcg 3720 tcctcccaac atggggcaat tgggcatacc catgttgtcacgtcactcag ctccgcgctc 3780 aacaccttct cgcgttggaa aacattagcg acatttacctggtgagcaat cagacatgcg 3840 acggctttag cctggcctcc ttaaattcac ctaagaatgggagcaaccag catgcaggaa 3900 aaggacaagc agcgaaaatt cacgccccct tgggaggtggcggcatatgc aaaggatagc 3960 actcccactc tactactggg tatcatatgc tgactgtatatgcatgagga tagcatatgc 4020 tacccggata cagattagga tagcatatac tacccagatatagattagga tagcatatgc 4080 tacccagata tagattagga tagcctatgc tacccagatataaattagga tagcatatac 4140 tacccagata tagattagga tagcatatgc tacccagatatagattagga tagcctatgc 4200 tacccagata tagattagga tagcatatgc tacccagatatagattagga tagcatatgc 4260 tatccagata tttgggtagt atatgctacc cagatataaattaggatagc atatactacc 4320 ctaatctcta ttaggatagc atatgctacc cggatacagattaggatagc atatactacc 4380 cagatataga ttaggatagc atatgctacc cagatatagattaggatagc ctatgctacc 4440 cagatataaa ttaggatagc atatactacc cagatatagattaggatagc atatgctacc 4500 cagatataga ttaggatagc ctatgctacc cagatatagattaggatagc atatgctatc 4560 cagatatttg ggtagtatat gctacccatg gcaacattagcccaccgtgc tctcagcgac 4620 ctcgtgaata tgaggaccaa caaccctgtg cttggcgctcaggcgcaagt gtgtgtaatt 4680 tgtcctccag atcgcagcaa tcgcgcccct atcttggcccgcccacctac ttatgcaggt 4740 attccccggg gtgccattag tggttttgtg ggcaagtggtttgaccgcag tggttagcgg 4800 ggttacaatc agccaagtta ttacaccctt attttacagtccaaaaccgc agggcggcgt 4860 gtgggggctg acgcgtgccc ccactccaca atttcaaaaaaaagagtggc cacttgtctt 4920 tgtttatggg ccccattggc gtggagcccc gtttaattttcgggggtgtt agagacaacc 4980 agtggagtcc gctgctgtcg gcgtccactc tctttccccttgttacaaat agagtgtaac 5040 aacatggttc acctgtcttg gtccctgcct gggacacatcttaataaccc cagtatcata 5100 ttgcactagg attatgtgtt gcccatagcc ataaattcgtgtgagatgga catccagtct 5160 ttacggcttg tccccacccc atggatttct attgttaaagatattcagaa tgtttcattc 5220 ctacactagt atttattgcc caaggggttt gtgagggttatattggtgtc atagcacaat 5280 gccaccactg aaccccccgt ccaaatttta ttctgggggcgtcacctgaa accttgtttt 5340 cgagcacctc acatacacct tactgttcac aactcagcagttattctatt agctaaacga 5400 aggagaatga agaagcaggc gaagattcag gagagttcactgcccgctcc ttgatcttca 5460 gccactgccc ttgtgactaa aatggttcac taccctcgtggaatcctgac cccatgtaaa 5520 taaaaccgtg acagctcatg gggtgggaga tatcgctgttccttaggacc cttttactaa 5580 ccctaattcg atagcatatg cttcccgttg ggtaacatatgctattgaat tagggttagt 5640 ctggatagta tatactacta cccgggaagc atatgctacccgtttagggt taacaagggg 5700 gccttataaa cactattgct aatgccctct tgagggtccgcttatcggta gctacacagg 5760 cccctctgat tgacgttggt gtagcctccc gtagtcttcctgggcccctg ggaggtacat 5820 gtcccccagc attggtgtaa gagcttcagc caagagttacacataaaggc aatgttgtgt 5880 tgcagtccac agactgcaaa gtctgctcca ggatgaaagccactcagtgt tggcaaatgt 5940 gcacatccat ttataaggat gtcaactaca gtcagagaacccctttgtgt ttggtccccc 6000 cccgtgtcac atgtggaaca gggcccagtt ggcaagttgtaccaaccaac tgaagggatt 6060 acatgcactg ccccgaatac aaaacaaaag cgctcctcgtaccagcgaag aaggggcaga 6120 gatgccgtag tcaggtttag ttcgtccggc ggcgggcggccgcaaggcgc gccggatcca 6180 caggacgggt gtggtcgcca tgatcgcgta gtcgatagtggctccaagta gcgaagcgag 6240 caggactggg cggcggccaa agcggtcgga cagtgctccgagaacgggtg cgcatagaaa 6300 ttgcatcaac gcatatagcg ctagatcctt gctagagtcgagatctgtcg agccatgtga 6360 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccgcgttgctggc gtttttccat 6420 aggctccgcc cccctgacga gcatcacaaa aatcgacgctcaagtcagag gtggcgaaac 6480 ccgacaggac tataaagata ccaggcgttt ccccctggaagctccctcgt gcgctctcct 6540 gttccgaccc tgccgcttac cggatacctg tccgcctttctcccttcggg aagcgtggcg 6600 ctttctcata gctcacgctg taggtatctc agttcggtgtaggtcgttcg ctccaagctg 6660 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcgccttatccgg taactatcgt 6720 cttgagtcca acccggtaag acacgactta tcgccactggcagcagccac tggtaacagg 6780 attagcagag cgaggtatgt aggcggtgct acagagttcttgaagtggtg gcctaactac 6840 ggctacacta gaaggacagt atttggtatc tgcgctctgctgaagccagt taccttcgga 6900 aaaagagttg gtagctcttg atccggcaaa caaaccaccgctggtagcgg tggttttttt 6960 gtttgcaagc agcagattac gcgcagaaaa aaaggatctcaagaagatcc tttgatcttt 7020 tctacggggt ctgacgctca gtggaacgaa aactcacgttaagggatttt ggtcatgaga 7080 ttatcaaaaa ggatcttcac ctagatcctt ttatcggtgtgaaataccgc acagatgcgt 7140 aaggagaaaa taccgcatca ggaaattgta agcgttaataattcagaaga actcgtcaag 7200 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcgataccgtaaa gcacgaggaa 7260 gcggtcagcc cattcgccgc caagctcttc agcaatatcacgggtagcca acgctatgtc 7320 ctgatagcgg tccgccacac ccagccggcc acagtcgatgaatccagaaa agcggccatt 7380 ttccaccatg atattcggca agcaggcatc gccatgggtcacgacgagat cctcgccgtc 7440 gggcatgctc gccttgagcc tggcgaacag ttcggctggcgcgagcccct gatgctcttc 7500 gtccagatca tcctgatcga caagaccggc ttccatccgagtacgtgctc gctcgatgcg 7560 atgtttcgct tggtggtcga atgggcaggt agccggatcaagcgtatgca gccgccgcat 7620 tgcatcagcc atgatggata ctttctcggc aggagcaaggtgagatgaca ggagatcctg 7680 ccccggcact tcgcccaata gcagccagtc ccttcccgcttcagtgacaa cgtcgagcac 7740 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagccgcgctgcct cgtcttgcag 7800 ttcattcagg gcaccggaca ggtcggtctt gacaaaaagaaccgggcgcc cctgcgctga 7860 cagccggaac acggcggcat cagagcagcc gattgtctgttgtgcccagt catagccgaa 7920 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaatccatcttgtt caatcatgcg 7980 aaacgatcct catcctgtct cttgatcaga gcttgatcccctgcgccatc agatccttgg 8040 cggcgagaaa gccatccagt ttactttgca gggcttgtcaaccttaccag ataaaagtgc 8100 tcatcattgg aaaacattca attcgtcgac ctcgaaattctaccgggtag gggaggcgct 8160 tttcccaagg cagtctggag catgcgcttt agcagccccgctgggcactt ggcgctacac 8220 aagtggcctc tggcctcgca cacattccac atccaccggtaggcgccaac cggctccgtt 8280 ctttggtggc cccttcgcgc caccttctac tcctcccctagtcaggaagt tcccccccgc 8340 cccgcanctc gcgtcgtgca ggacgtgaca aatggaaatagcacgtctca ctagtctcgt 8400 gcagatggac aagcaccgct gagcaatgga gcgggtaggcctttggggca gcggccaata 8460 gcagctttgc tccttcgctt tctgggctca gaggctggnaaggggtgggt ccgggggcgg 8520 gctcaggggc gggctcaggg gcggggcggg cgcccgaaggtcctccggag gcccggcatt 8580 ctgcacgctt caaaagcgca cgtctgccgc gctgttctcctcttcctcat ctccgggcct 8640 ttcgacctgc atccatctag atctcgagca gctgaagcttaccatgaccg agtacaagcc 8700 cacggtgcgc ctcgccaccc gcgacgacgt cccccgggccgtacgcaccc tcgccgccgc 8760 gttcgccgac taccccgcca cgcgccacac cgtcgacccggaccgccaca tcgagcgggt 8820 caccgagctg caagaactct tcctcacgcg cgtcgggctcgacatcggca aggtgtgggt 8880 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccggagagcgtcg aagcgggggc 8940 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagcggttcccggc tggccgcgca 9000 gcaacagatg gaaggcctcc tggcgccgca ccgggcccaaggagcccgcg tggttccttg 9060 gcccaccgtc gggcgtcttc gcccgaccac cagggcaagggtctggcaag cgccgtcgtg 9120 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccgccttcctgga gacctccgcg 9180 ccccgcaacc tccccttcta cgagcggctc ggcttcaccgtcaccgccga cgtcgaggtg 9240 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccggtgcctgacg cccgccccac 9300 gacccgcagc gcccgaccga aaggagcgca cgaccccatgcatcgatggc actgggcagg 9360 taagtatcaa ggttagcggc cgctaacctg gttgctgactaattgagatg catgctttgc 9420 atacttctgc ctgctgggga gcctggggac tttccacaccctaactgaca cacattccac 9480 agctggttct ttccgcctca gaaggtacac aggcgaaattgtaagcgtta atattttgtt 9540 aaaattcgcg ttaaattttt gttaaatcag ctcattttttaaccaatagg ccgaaatcgg 9600 caaaatccct tataaatcaa aagaatagac cgagatagggttgagtgttg ttccagtttg 9660 gaacaagagt ccactattaa agaacgtgga ctccaacgtcaaagggcgaa aaaccgtcta 9720 tcagggcgat ggcccac 9737 29 12 DNA ArtificialSequence Description of Artificial Sequence Vector Promoter 29acccaggtga tg 12 30 15 DNA Artificial Sequence Description of ArtificialSequence Vector Promoter 30 accatgcagg tgatg 15 31 16 DNA ArtificialSequence Description of Artificial Sequence Vector Promoter 31accatggcag gtgatg 16 32 17 DNA Artificial Sequence Description ofArtificial Sequence Vector Promoter 32 accatgggca ggtgatg 17 33 10 DNAArtificial Sequence Description of Artificial Sequence Vector 33aaaaaaaaaa 10

What is claimed is:
 1. A eukaryotic cell in vitro comprising a vectorconstruct comprising a selectable marker and a transcriptionalregulatory sequence that is operably linked to an exon comprising atranslational start codon, a secretion signal sequence, and an epitopetag sequence, said exon defined at the 3′ end by an unpaired splicedonor site, wherein said vector construct does not contain a targetingsequence.
 2. A eukaryotic cell in vitro comprising a vector constructcomprising a transcriptional regulatory sequence that is operably linkedto an exon comprising a translational start codon, a secretion signalsequence, and an epitope tag sequence, said exon defined at the 3′ endby an unpaired splice donor site, wherein said vector construct does notcontain a targeting sequence, and wherein said construct furthercomprises one or more amplifiable markers.
 3. The cell of claim 1,wherein said vector construct has integrated into the genome of saidcell.
 4. The cell of claim 2, wherein said vector construct hasintegrated into the genome of said cell.
 5. A eukaryotic cell in vitrocomprising a vector construct, said vector construct comprising atranscriptional regulatory sequence that is operably linked to an exoncomprising a translational start codon, a secretion signal sequence, andan epitope tag sequence, said exon defined at the 3′ end by an unpairedsplice donor site, wherein said vector construct has integrated, bynon-homologous recombination, into the genome of said cell, wherein anendogenons gene in said genome is over-expressed in said cell byup-regulation of the gene in said cell by said transcriptionalregulatory sequence on said vector construct.
 6. The cell of claim 1,wherein said cell is an isolated cell.
 7. The cell of claim 2, whereinsaid cell is an isolated cell.
 8. A method for making a eukaryotic cellcomprising a vector constructs said method comprising introducing into aeukaryotic cell in vitro a vector construct comprising a selectablemarker and a transcriptional regulatory sequence that is operably linkedto an exon comprising a translational start codon, a secretion signalsequence, and an epitope tag sequence, said exon defined at the 3′ endby an unpaired splice donor site, wherein said vector construct does notcontain a targeting sequence.
 9. A method for producing an expressionproduct of an endogenous cellular gene or portion thereof comprising:(a) introducing into a eukaryotic cell in vitro a vector constructcomprising a transcriptional regulatory sequence that is operably linkedto an exon comprising a translational start codon, a secretion signalsequence, and an epitope tag sequence, said exon defined at the 3′ endby an unpaired splice donor site; (b) maintaining said cell underconditions suitable for integrating said vector construct into thegenome of said cell by non-homologous recombination such that thetranscriptional regulatory sequence is operably linked to saidendogenous cellular gene; and (c) maintaining said cell produced in step(b) under conditions suitable for over-expressing, by means of saidintegrated transcriptional regulatory sequence, said endogenous cellulargene or portion thereof in said cell thereby producing said expressionproduct.
 10. The method of claim 9, further comprising isolating saidexpression product produced by said cell following over-expression. 11.A eukaryotic cell library comprising a collection of tells in vitrotransformed with a vector construct comprising a transcriptionalregulatory sequence that is operably linked to an exon comprising atranslational start codon, a secretion signal sequence, and an epitopetag sequence, said exon defined at the 3′ end by an unpaired splicedonor site, wherein said vector construct is integrated into the genomesof said cells by non-homologous recombination.
 12. A method of obtaininga gene product from a library of eukaryotic cells in vitro, said geneproduct being produced by up-regulation of a gene by a vector constructcomprising a transcriptional regulatory sequence that is operably linkedto an exon comprising a translational start codon, a secretion signalsequence, and an epitope tag sequence, said exon defined at the 3′ endby an unpaired splice donor site, said method comprising screening thelibrary of claim 11 for expression of said gene product, selecting fromsaid library a cell that over-expresses said gene product, and obtainingsaid gene product produced by said selected cell.
 13. The method ofclaim 9, further comprising isolating said produced expression product.14. A method for producing an expression product of an endogenous genein a cell comprising: (a) introducing into at least one eukaryotic cellin vitro a vector construct comprising a transcriptional regulatorysequence that is operably linked to an exon comprising a translationalstart codon, a secretion signal sequence, and an epitope tag sequence,said exon defined at the 3′ end by an unpaired splice donor site; (b)maintaining said cell under conditions suitable for integrating saidvector construct into the genome of said cell by non-homologousrecombination such that the transcriptional regulatory sequence isoperably linked to said endogenous cellular gene; (c) maintaining saidcell produced in step (b) under conditions suitable for over-expressingan endogenous gene or a portion thereof in said cell by up-regulation ofsaid gene in said cell by said transcriptional regulatory sequence; and(d) culturing said cell in serum-free medium, resulting in production ofsaid expression product.
 15. A method of identifying a protein productover-expressed by an endogenous gene, said method comprising: (a)introducing into at least one eukaryotic cell in vitro a vectorconstruct comprising a transcriptional regulatory sequence that isoperably linked to an exon comprising a translational start codon, asecretion signal sequence, and an epitope tag sequence, said exondefined at the 3′ end by an unpaired splice donor site; (b) maintainingsaid cell under conditions suitable for integrating said vectorconstruct into the genome of said cell by non-homologous recombinationsuch that the transcriptional regulatory sequence is operably linked tosaid endogenous gene; (c) culturing said cell in serum-free medium underconditions that allow over-expression of said endogenous gene or aportion thereof in said cell by up-regulation of said endogenous gene insaid cell by said transcriptional regulatory sequence, thereby producingcell-conditioned media; and (d) screening said cell-conditioned mediafor the presence of the protein product of over-expression of saidendogenous gene or portion thereof, thereby identifying said proteinproduct.
 16. The method of claim 15, further comprising concentratingsaid cell-conditioned media prior to screening in (d).
 17. The method ofany of claims 10 or 14-16, wherein said endogenous gene encodes atransmembrane protein, and wherein said method includes an assay forsaid transmembrane protein.
 18. A method for expressing a gene orportion thereof, comprising: (a) obtaining genomic DNA, comprising oneor more genes, or a portion of said one or more genes, from one or moreeukaryotic cells; (b) introducing into a cell in vitro said genomic DNAand a vector, said vector comprising a selectable marker and atranscriptional regulatory sequence that is operably linked to an exoncomprising a translational start codon, a secretion signal sequence, andan epitope tag sequence, said exon being defined at the 3′ end by anunpaired splice donor site; (c) selecting for a cell containing saidgenomic DNA and said vector; and (d) culturing said cell underconditions suitable for expression of said gene or portion thereof,wherein said gene or portion thereof is present on said genomic DNA andsaid expression results from the operable linkage between saidtranscriptional regulatory sequence on said vector construct and saidgene or portion thereof.
 19. A method for activating expression from anendogenous gene comprising: (a) introducing into a eukaryoptic cell invitro a vector construct comprising a transcriptional regulatorysequence that is operably linked to an exon comprising a translationalstart codon, a secretion signal sequence, and an epitope tag sequence,said exon defined at the 3′ end by an unpaired splice donor site; (b)introducing DNA breaks in the chromosome of said cell prior to orfollowing introduction of said vector construct; and (c) maintainingsaid cell under conditions suitable for integrating said vectorconstruct, by non-homologous recombination, into said chromosome so asto result in the formation of an operable linkage between said vectorconstruct and said endogenous gene, whereby said endogenous gene isactivated by one or more vector construct-encoded nucleotide sequences.20. The method of claim 19 further comprising isolating said cell afterstep (c).
 21. The method of any of claims 9, 12, 18 and 19, wherein saidvector construct further comprises one or more amplifiable markers. 22.The method of any of claims 9, 12, 14, 15, 18 and 19, wherein saidtranscriptional regulatory sequence comprises a promoter.
 23. The methodof claim 22, wherein said promoter is a viral promoter.
 24. The methodof claim 23, wherein said viral promoter is the cytomegalovirusimmediate early promoter.
 25. The method of claim 22, wherein saidpromoter is a non-viral promoter.
 26. The method of claim 22, whereinsaid promoter is an inducible promoter.
 27. The method of any of claims9, 12, 18 and 19, wherein said cell is a mammalian cell.
 28. The methodof claim 27 wherein said mammalian cell is selected from the groupconsisting of human, murine, rat, bovine, porcine, and ovine cells. 29.The method of claim 28 wherein said mammalian cell is a human cell. 30.The method of claim 28 wherein said mammalian cell is a murine cell. 31.The method of claim 18 wherein said vector construct comprises a viralorigin of replication.
 32. The method of claim 31 wherein said viralorigin of replication is from Epstein Barr virus.
 33. The method ofclaim 31 wherein said viral origin of replication is from SV40.
 34. Themethod of claim 18 wherein said vector construct comprises transpositionsignals.
 35. The method of claim 18 wherein the operably linked vectorand genomic DNA is integrated into the genome of said cell.
 36. Themethod of claim 31 wherein said vector and said genomic DNA are episomalin said cell.
 37. The method of claim 31 wherein said vector constructfurther comprises transposition signals.
 38. The method of any of claims9, 12, 18 and 19, wherein said cell in vitro is in culture.
 39. The cellof claim 5, wherein said vector construct further comprises one or moreamplifiable markers.
 40. The cell of any of claims 1, 2, or 5, whereinsaid transcriptional regulatory sequence comprises a promoter.
 41. Thecell of claim 40, wherein said promoter is a viral promoter.
 42. Thecell of claim 41, wherein said viral promoter is the cytomegalovirusimmediate early promoter.
 43. The cell of claim 40, wherein saidpromoter is a non-viral promoter.
 44. The cell of claim 40, wherein saidpromoter is an inducible promoter.
 45. The cell of any of claims 1, 2,or 5, wherein said cell is a mammalian cell.
 46. The cell of claim 45wherein said mammalian cell is selected from the group consisting ofhuman, murine, rat, bovine, porcine, and ovine cells.
 47. The cell ofclaim 46 wherein said mammalian cell is a human cell.
 48. The cell ofclaim 47 wherein said mammalian cell is a murine cell.
 49. The method ofclaim 18 wherein the operably linked vector and genomic DNA isextrachromosomal in said cell.